Regeneration Of Corticospinal Axons Research Articles

Deep scRNA sequencing reveals a broadly applicable Regeneration Classifier and implicates antioxidant response in corticospinal axon regeneration

Despite substantial progress in understanding the biology of axon regeneration in the CNS, our ability to promote regeneration of the clinically important corticospinal tract (CST) after spinal cord injury remains limited. To understand regenerative heterogeneity, we conducted patch-based single-cell RNA sequencing on rare regenerating CST neurons at high depth following PTEN and SOCS3 deletion. Supervised classification with Garnett gave rise to a Regeneration Classifier, which can be broadly applied to predict the regenerative potential of diverse neuronal types across developmental stages or after injury. Network analyses highlighted the importance of antioxidant response and mitochondrial biogenesis. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Our data demonstrate a universal transcriptomic signature underlying the regenerative potential of vastly different neuronal populations and illustrate that deep sequencing of only hundreds of phenotypically identified neurons has the power to advance regenerative biology.

Differences in Anatomical Outcomes Between Early Chronic and Far Chronic Time-Points After Transplantation of Spinal Cord Neural Progenitor Cells in Mice.

Spinal cord injury (SCI) affects millions of people worldwide. Neural progenitor cell (NPC) transplantation is a promising treatment for regenerating lost spinal cord tissue and restoring neurological function after SCI. We conducted a literature search and found that less than a quarter of experimental rodent cell and tissue transplantation studies have investigated anatomical outcomes at longer than 4 months post-transplantation. This is a critical topic to investigate, given that stem and progenitor cell therapies would need to remain in place throughout the lifetime of an individual. We sought to determine how commonly assessed anatomical outcomes evolve between early and far chronic time-points post-NPC transplantation. At either 8 weeks or 26 weeks following transplantation of NPCs into sites of cervical SCI, we evaluated graft neuronal density, astroglial cell density, graft axon outgrowth, and regeneration of host axon populations into grafts in male and female mice. We found that graft neuronal density does not change over time, but the numbers of graft-associated astrocytes and glial fibrillary acidic protein intensity is significantly increased in the far chronic phase compared with the early chronic time-point. In addition, graft axon outgrowth was significantly decreased at 26 weeks post-transplantation compared with 8 weeks post-transplantation. In contrast, corticospinal axon regeneration into grafts was not diminished over time, but rather increased significantly from early to far chronic periods. Interestingly, we found that graft neuronal density is significantly influenced by sex of the host animal, suggesting that sex-dependent processes may shape graft composition over time. Collectively, these results demonstrate that NPC transplants are dynamic and that commonly assessed outcome measures associated with graft efficacy evolve over the weeks to months post-transplantation into the spinal cord.

Dr. Xiao-Ming Xu: A True Gentleman and Scholar in the Field of Spinal Cord Injury and Schwann Cell Biology.

Dr. Xiao-Ming Xu: A True Gentleman and Scholar in the Field of Spinal Cord Injury and Schwann Cell Biology.

Rehabilitation combined with neural progenitor cell grafts enables functional recovery in chronic spinal cord injury.

We reported previously that neural progenitor cell (NPC) grafts form neural relays across sites of subacute spinal cord injury (SCI) and support functional recovery. Here, we examine whether NPC grafts after chronic delays also support recovery and whether intensive rehabilitation further enhances recovery. One month after severe bilateral cervical contusion, rats received daily intensive rehabilitation, NPC grafts, or both rehabilitation and grafts. Notably, only the combination of rehabilitation and grafting significantly improved functional recovery. Moreover, improved functional outcomes were associated with a rehabilitation-induced increase in host corticospinal axon regeneration into grafts. These findings identify a critical and synergistic role of rehabilitation and neural stem cell therapy in driving neural plasticity to support functional recovery after chronic and severe SCI.

Rabphilin3A reduces integrin-dependent growth cone signaling to restrict axon regeneration after trauma

Neural repair after traumatic spinal cord injury depends upon the restoration of neural networks via axonal sprouting and regeneration. Our previous genome wide loss-of-function screen identified Rab GTPases as playing a prominent role in preventing successful axon sprouting and regeneration. Here, we searched for Rab27b interactors and identified Rabphilin3A as an effector within regenerating axons. Growth cone Rabphilin3a colocalized and physically associated with integrins at puncta in the proximal body of the axonal growth cone. In regenerating axons, loss of Rabphilin3a increased integrin enrichment in the growth cone periphery, enhanced focal adhesion kinase activation, increased F-actin-rich filopodial density and stimulated axon extension. Compared to wild type, mice lacking Rabphilin3a exhibited greater regeneration of retinal ganglion cell axons after optic nerve crush as well as greater corticospinal axon regeneration after complete thoracic spinal cord crush injury. After moderate spinal cord contusion injury, there was greater corticospinal regrowth in the absence of Rph3a. Thus, an endogenous Rab27b - Raphilin3a pathway limits integrin action in the growth cone, and deletion of this monomeric GTPase pathway permits reparative axon growth in the injured adult mammalian central nervous system.

PTEN Blocking Stimulates Corticospinal and Raphespinal Axonal Regeneration and Promotes Functional Recovery After Spinal Cord Injury.

The long-term disabilities associated with spinal cord injury (SCI) are primarily due to the absence of robust neuronal regeneration and functional plasticity. The inability of the axon to regenerate after SCI is contributed by several intrinsic factors that trigger a cascade of molecular growth program and modulates axonal sprouting. Phosphatase and tensin homolog (PTEN) is one of the intrinsic factors contributing to growth failure after SCI, however, the underlying mechanism is not well known. Here, we developed a novel therapeutic approach for treating SCI by suppressing the action of PTEN in a mouse model of hemisection SCI. We have used a novel peptide, PTEN antagonistic peptide (PAP) to block the critical domains of PTEN to demonstrate its ability to potentially promote axon growth. PAP treatment not only enhanced regeneration of corticospinal axons into the caudal spinal cord but also promoted the regrowth of descending serotonergic axons in SCI mice. Furthermore, expression levels of p-mTOR, p-S6, p-Akt, p-Erk, p-GSK, p-PI3K downstream of PTEN signaling pathway were increased significantly in the spinal cord of SCI mice systemically treated with PAP than control TAT peptide-treated mice. Our novel strategy of administering deliverable compounds postinjury may facilitate translational feasibility for central nervous system injury.

Glial Metabolic Rewiring Promotes Axon Regeneration and Functional Recovery in the Central Nervous System

Axons in the mature central nervous system (CNS) fail to regenerate after axotomy, partly due to the inhibitory environment constituted by reactive glial cells producing astrocytic scars, chondroitin sulfate proteoglycans, and myelin debris. We investigated this inhibitory milieu, showing that it is reversible and depends on glial metabolic status. We show that glia can be reprogrammed to promote morphological and functional regeneration after CNS injury in Drosophila via increased glycolysis. This enhancement is mediated by the glia derived metabolites: L-lactate and L-2-hydroxyglutarate (L-2HG). Genetically/pharmacologically increasing or reducing their bioactivity promoted or impeded CNS axon regeneration. L-lactate and L-2HG from glia acted on neuronal metabotropic GABAB receptors to boost cAMP signaling. Local application of L-lactate to injured spinal cord promoted corticospinal tract axon regeneration, leading to behavioral recovery in adult mice. Our findings revealed a metabolic switch to circumvent the inhibition of glia while amplifying their beneficial effects for treating CNS injuries.

Upregulating Lin28a Promotes Axon Regeneration in Adult Mice with Optic Nerve and Spinal Cord Injury

Severed CNS axons fail to regenerate in adult mammals and there are no effective regenerative strategies to treat patients with CNS injuries. Several genes, including phosphatase and tensin homolog (PTEN) and Krüppel-like factors, regulate intrinsic growth capacity of mature neurons. The Lin28 gene is essential for cell development and pluripotency in worms and mammals. In this study, we evaluated the role of Lin28a in regulating regenerative capacity of diverse populations of CNS neurons in adult mammals. Using a neuron-specific Thy1 promoter, we generated transgenic mice that overexpress Lin28a protein in multiple populations of projection neurons, including corticospinal tracts and retinal ganglion cells. We demonstrate that upregulation of Lin28a in transgenic mice induces significant long distance regeneration of both corticospinal axons and the optic nerve in adult mice. Importantly, overexpression of Lin28a by post-injury treatment with adeno-associated virus type 2 (AAV2) vector stimulates dramatic regeneration of descending spinal tracts and optic nerve axons after lesions. Upregulation of Lin28a also enhances activity of the Akt signaling pathway in mature CNS neurons. Therefore, Lin28a is critical for regulating growth capacity of multiple CNS neurons and may become an important molecular target for treating CNS injuries.

Injured adult neurons regress to an embryonic transcriptional growth state.

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.

Regeneration of Corticospinal Axons into Neural Progenitor Cell Grafts After Spinal Cord Injury.

Spinal cord injuries leave patients with lifelong paralysis. To date, there are no therapies that promote the critical step required for the recovery of voluntary motor function: corticospinal axon regeneration. Spinal cord-derived neural progenitor cell (NPC) grafts integrate into the injured host spinal cord, enable robust corticospinal axon regeneration, and restore forelimb function following spinal cord injury in rodents. Consequently, engineered stem cell differentiation and transplantation techniques harbor promising potential for the design and implementation of therapies promoting corticospinal axon regeneration. However, in order to optimize the outcome of clinical trials, it is critical to fully understand the cellular and molecular mechanisms underlying this regeneration. Our recent study highlights the unexpected intrinsic potential of corticospinal neurons to regenerate and allows us to investigate new hypotheses exploiting this newly discovered potential.

BMP4/Smad1 Signalling Promotes Spinal Dorsal Column Axon Regeneration and Functional Recovery After Injury

Signalling through the BMP4/Smad1 pathway promotes corticospinal tract axon regeneration and functional recovery in mice. However, unlike humans and rats, mice do not cavitate. Here, we investigated if activation of the BMP4/Smad1 pathway promotes axon regeneration and functional recovery in a rat model that cavitates. We show that dorsal root ganglion neurons (DRGN) in injury models, including the non-regenerating dorsal column (DC) and the regenerating sciatic nerve (SN) crush and preconditioning (p) SN + DC (pSN + DC) paradigms, regulate the BMP4/Smad1 signalling pathway. For example, mRNA expression of positive regulators of the BMP4/Smad1 pathway was highly up-regulated whilst negative regulators were significantly down-regulated in DRGN in the regenerating SN and pSN + DC models compared to non-regenerating DC models, matched by concomitant changes in protein expression detected in DRGN by immunohistochemistry. BMP4 peptide promoted significant DRGN survival and disinhibited neurite outgrowth in vitro, whilst AAV-BMP4 delivery in vivo stimulated DC axon regeneration and functional recovery in a model that cavitates. Our results show that activation of the BMP4/Smad1 pathway is a potential therapeutic target in the search for axon regenerative signalling pathways in the CNS.

Regenerating Corticospinal Axons Innervate Phenotypically Appropriate Neurons within Neural Stem Cell Grafts

SUMMARYNeural progenitor cell grafts form new relays across sites of spinal cord injury (SCI). Using a panel of neuronal markers, we demonstrate that spinal neural progenitor grafts to sites of rodent SCI adopt diverse spinal motor and sensory interneuronal fates, representing most neuronal subtypes of the intact spinal cord, and spontaneously segregate into domains of distinct cell clusters. Host corticospinal motor axons regenerating into neural progenitor grafts innervate appropriate pre-motor interneurons, based on trans-synaptic tracing with herpes simplex virus. A human spinal neural progenitor cell graft to a non-human primate also received topographically appropriate corticospinal axon regeneration. Thus, grafted spinal neural progenitor cells give rise to a variety of neuronal progeny that are typical of the normal spinal cord; remarkably, regenerating injured adult corticospinal motor axons spontaneously locate appropriate motor domains in the heterogeneous, developing graft environment, without a need for additional exogenous guidance.

Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1.

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

Reducing Pericyte-Derived Scarring Promotes Recovery after Spinal Cord Injury

SummaryCNS injury often severs axons. Scar tissue that forms locally at the lesion site is thought to block axonal regeneration, resulting in permanent functional deficits. We report that inhibiting the generation of progeny by a subclass of pericytes led to decreased fibrosis and extracellular matrix deposition after spinal cord injury in mice. Regeneration of raphespinal and corticospinal tract axons was enhanced and sensorimotor function recovery improved following spinal cord injury in animals with attenuated pericyte-derived scarring. Using optogenetic stimulation, we demonstrate that regenerated corticospinal tract axons integrated into the local spinal cord circuitry below the lesion site. The number of regenerated axons correlated with improved sensorimotor function recovery. In conclusion, attenuation of pericyte-derived fibrosis represents a promising therapeutic approach to facilitate recovery following CNS injury.

Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration.

The corticospinal tract (CST) is the most important motor system in humans, yet robust regeneration of this projection after spinal cord injury (SCI) has not been accomplished. In rodent models of SCI, we report robust corticospinal axon regeneration, functional synapse formation and improved skilled forelimb function after grafting multipotent neural progenitor cells into sites of spinal cord injury. Corticospinal regeneration requires that grafts are driven toward caudalized (spinal cord), rather than rostralized, fates. Fully mature caudalized neural grafts also support corticospinal regeneration. Moreover, corticospinal axons can emerge from neural grafts and regenerate beyond the lesion, potentially related to attenuation of the glial scar. Rodent corticospinal axons also regenerate into human donor grafts of caudal spinal cord identity. Collectively, these findings indicate that spinal cord “replacement” with homologous neural stem cells enables robust regeneration of the corticospinal projection within and beyond spinal cord lesion sites, achieving a major unmet goal of spinal cord injury research and opening new possibilities for translation.

Biomaterial bridges enable regeneration and re-entry of corticospinal tract axons into the caudal spinal cord after SCI: Association with recovery of forelimb function

Severed axon tracts fail to exhibit robust or spontaneous regeneration after spinal cord injury (SCI). Regeneration failure reflects a combination of factors, including the growth state of neuronal cell bodies and the regeneration-inhibitory environment of the central nervous system. However, while spared circuitry can be retrained, target reinnervation depends on longitudinally directed regeneration of transected axons. This study describes a biodegradable implant using poly(lactide-co-glycolide) (PLG) bridges as a carrier scaffold to support regeneration after injury. In order to detect regeneration of descending neuronal tracts into the bridge, and beyond into intact caudal parenchyma, we developed a mouse cervical implantation model and employed Crym:GFP transgenic mice. Characterization of Crym:GFP mice revealed that descending tracts, including the corticospinal tract, were labeled by green fluorescent protein (GFP), while ascending sensory neurons and fibers were not. Robust co-localization between GFP and neurofilament-200 (NF-200) as well as GFP and GAP-43 was observed at both the rostral and caudal bridge/tissue interface. No evidence of similar regeneration was observed in mice that received gelfoam at the lesion site as controls. Minimal co-localization between GFP reporter labeling and macrophage markers was observed. Taken together, these data suggest that axons originating from descending fiber tracts regenerated, entered into the PLG bridge at the rostral margin, continued through the bridge site, and exited to re-enter host tissue at the caudal edge of the intact bridge. Finally, regeneration through implanted bridges was associated with a reduction in ipsilateral forelimb errors on a horizontal ladder task.

Effects of PTEN and Nogo Codeletion on Corticospinal Axon Sprouting and Regeneration in Mice.

Axons in the adult CNS have poor ability to grow after injury, impeding functional recovery in patients of spinal cord injury. This has been attributed to both a developmental decline in neuron-intrinsic growth ability and the presence of extrinsic growth inhibitors. We previously showed that genetic deletion of Nogo, an extrinsic inhibitor, promoted axonal sprouting from uninjured corticospinal tract (CST) neurons but not regeneration from injured CST neurons, whereas genetic deletion of PTEN, an intrinsic inhibitor, promoted both CST sprouting and regeneration. Here we test the hypothesis that combining an elevation of neuron-intrinsic growth ability and a reduction of extrinsic growth inhibition by genetic codeletion of PTEN and Nogo may further improve injury-induced axonal growth. In an apparent paradox, additionally deleting Nogo further enhanced CST regeneration but not sprouting in PTEN-deleted mice. Enhanced CST regeneration and sprouting in PTEN and PTEN/Nogo-deleted mice were associated with no or only temporary improvement in functional recovery. Our data illustrate that neuron-intrinsic and -extrinsic factors regulate axon regeneration and sprouting in complex ways and provide proof-of-principle evidence that targeting both can further improve regeneration. Neuron-intrinsic growth ability is an important determinant of neuronal responsiveness to changes in extrinsic growth inhibition, such that an elevated intrinsic growth state is a prerequisite for reducing extrinsic inhibition to take effect on CST regeneration. Meanwhile, additional strategies are required to unleash the full potential for functional recovery with enhanced axon regeneration and/or sprouting.

Improved axonal regeneration after spinal cord injury in mice with conditional deletion of ephrin B2 under the GFAP promoter

Spinal cord injury (SCI) initiates a cascade of processes that ultimately form a nonpermissive environment for axonal regeneration. Emerging evidence suggests that regenerative failure may be due in part to inhibitory factors expressed by reactive spinal cord glial cells and meningeal fibroblasts, such as the Eph receptor protein-tyrosine kinases and their corresponding ligands (ephrins). Here we sought to assess the role of ephrin B2, an inhibitory axonal guidance molecule, as an inhibitor of the recovery process following SCI. To determine the extent of ephrin B2 involvement in axonal regenerative failure, a SCI model was performed on a conditional ephrin B2 knockout mouse strain (ephrin B2−/−), in which the ephrin B2 gene was deleted under the GFAP promoter . The expression of ephrin B2 was significantly decreased in astrocytes of injured and uninjured ephrin B2−/− mice compared to wild-type mice. Notably, in the ephrin B2−/− mice, the deletion of ephrin B2 reduced astrogliosis, and accelerated motor function recovery after SCI. Anterograde axonal tracing on a hemisection model of SCI further showed that ephrin B2−/− mice exhibited increased regeneration of injured corticospinal axons and a reduced glial scar, when compared to littermate controls exposed to similar injury. These results were confirmed by an in vitro neurite outgrowth assay and ephrin B2 functional blockage, which showed that ephrin B2 expressed on astrocytes inhibited axonal growth. Combined these findings suggest that ephrin B2 ligands expressed by reactive astrocytes impede the recovery process following SCI.

Lentiviral vectors express chondroitinase ABC in cortical projections and promote sprouting of injured corticospinal axons

Several diseases and injuries of the central nervous system could potentially be treated by delivery of an enzyme, which might most effectively be achieved by gene therapy. In particular, the bacterial enzyme chondroitinase ABC is beneficial in animal models of spinal cord injury. We have adapted the chondroitinase gene so that it can direct secretion of active chondroitinase from mammalian cells, and inserted it into lentiviral vectors. When injected into adult rat brain, these vectors lead to extensive secretion of chondroitinase, both locally and from long-distance axon projections, with activity persisting for more than 4 weeks. In animals which received a simultaneous lesion of the corticospinal tract, the vector reduced axonal die-back and promoted sprouting and short-range regeneration of corticospinal axons. The same beneficial effects on damaged corticospinal axons were observed in animals which received the chondroitinase lentiviral vector directly into the vicinity of a spinal cord lesion.

Enhanced regenerative axon growth of multiple fibre populations in traumatic spinal cord injury following scar‐suppressing treatment

We analysed the effect of scar-suppressing treatment (anti-scarring treatment; AST) on augmenting axonal regeneration of various neuronal populations following spinal cord injury (SCI) in adult rat. AST included local iron chelator (2,2'-dipyridine-5,5'-dicarboxylic acid) injection and 8-bromo-cyclic adenosine monophosphate application to the lesion core. In previous studies, this treatment promoted long-distance regeneration of cut corticospinal tract axons, neuroprotection of projecting cortical neurons and functional improvement of treated rats [N. Klapka et al. (2005)Eur. J. Neurosci., 22, 3047-3058]. Treatment yielded significantly enhanced regrowth of descending serotonergic (5-HT), catecholaminergic (tyrosine hydroxylase; TH), corticospinal and rubrospinal axons into the lesion zone, as assessed by anterograde tracing and immunohistochemistry followed by quantification of axon profiles at 5 and 12 weeks post-injury. In addition, the determination of axons crossing the proximal borderline from uninjured tissue into fibrous scar area revealed a significant AST-promoted increase of intersecting fibres for 5-HT, TH and calcitonin gene-related peptide containing ascending sensory fibres. For a prolonged time period after lesion, the delayed (secondary) scar developing in treated rats is significantly more permeable for all analysed axon tracts than the initial (primary) scar forming in injured control animals lacking treatment. Furthermore, enhanced outgrowth of descending axons from fibrous scar into distal healthy spinal tissue was achieved in treated animals, and is in line with previous functional studies [S. Hermanns et al. (2001) Restor. Neurol. Neurosci., 19,139-148; N. Klapka et al. (2005)Eur. J. Neurosci., 22, 3047-3058]. Our findings indicate that AST exerts a prolonged beneficial effect on fibrous scarring allowing enhanced axonal regrowth of different fibre tracts in SCI regardless of their distinct regenerative demands.