Blockade of Rho-associated kinase prevents inhibition of axon regeneration of peripheral nerves induced by anti-ganglioside antibodies

Anti-ganglioside antibodies are associated with delayed/poor clinical recovery in Guillain-Barrèsyndrome,mostly related to halted axon regeneration.Cross-linking of cell surface gangliosides by anti-ganglioside antibodies triggers inhibition of nerve repair in in vitro and in vivo paradigms of axon regeneration.These effects involve the activation of the small GTPase Rho A/ROCK signaling pathways,which negatively modulate growth cone cytoskeleton,similarly to well stablished inhibitors of axon regeneration described so far.The aim of this work was to perform a proof of concept study to demonstrate the effectiveness of Y-27632,a selective pharmacological inhibitor of ROCK,in a mouse model of axon regeneration of peripheral nerves,where the passive immunization with a monoclonal antibody targeting gangliosides GD1a and GT1b was previously reported to exert a potent inhibitory effect on regeneration of both myelinated and unmyelinated fibers.Our results demonstrate a differential sensitivity of myelinated and unmyelinated axons to the pro-regenerative effect of Y-27632.Treatment with a total dosage of 9 mg/kg of Y-27632 resulted in a complete prevention of anti-GD1a/GT1b monoclonal antibody-mediated inhibition of axon regeneration of unmyelinated fibers to skin and the functional recovery of mechanical cutaneous sensitivity.In contrast,the same dose showed toxic effects on the regeneration of myelinated fibers.Interestingly,scale down of the dosage of Y-27632 to 5 mg/kg resulted in a significant although not complete recovery of regenerated myelinated axons exposed to anti-GD1a/GT1b monoclonal antibody in the absence of toxicity in animals exposed to only Y-27632.Overall,these findings confirm the in vivo participation of Rho A/ROCK signaling pathways in the molecular mechanisms associated with the inhibition of axon regeneration induced by anti-GD1a/GT1b monoclonal antibody.Our findings open the possibility of therapeutic pharmacological intervention targeting Rho A/Rock pathway in immune neuropathies associated with the presence of anti-ganglioside antibodies and delayed or incomplete clinical recovery after injury in the peripheral nervous system.

Phosphorylated S6K1 and 4E-BP1 play different roles in constitutively active Rheb-mediated retinal ganglion cell survival and axon regeneration after optic nerve injury

Ras homolog enriched in brain(Rheb) is a small GTPase that activates mammalian target of rapamycin complex 1(mTORC1).Previous studies have shown that constitutively active Rheb can enhance the regeneration of sensory axons after spinal cord injury by activating downstream effectors of mTOR.S6K1 and4E-BP1 are important downstream effectors of mTORC1.In this study,we investigated the role of Rheb/mTOR and its downstream effectors S6K1 and 4E-BP1in the protection of retinal ganglion cells.We transfected an optic nerve crush mouse model with adeno-associated viral 2-mediated constitutively active Rheb and observed the effects on retinal ganglion cell survival and axon regeneration.We found that overexpression of constitutively active Rheb promoted survival of retinal ganglion cells in the acute(14 days) and chronic(21 and 42 days) stages of injury.We also found that either co-expression of the dominant-negative S6K1mutant or the constitutively active 4E-BP1 mutant together with constitutively active Rheb markedly inhibited axon regeneration of retinal ganglion cells.This suggests that mTORC1-mediated S6K1 activation and 4E-BP1 inhibition were necessary components for constitutively active Rheb-induced axon regeneration.However,only S6K1 activation,but not 4E-BP1 knockdown,induced axon regeneration when applied alone.Furthermore,S6K1 activation promoted the survival of retinal ganglion cells at 14 days post-injury,whereas 4E-BP1 knockdown unexpectedly slightly decreased the survival of retinal ganglion cells at 14 days postinjury.Ove rexpression of constitutively active 4E-BP1 increased the survival of retinal ganglion cells at 14 days post-injury.Likewise,co-expressing constitutively active Rheb and constitutively active 4E-BP1 markedly increased the survival of retinal ganglion cells compared with overexpression of constitutively active Rheb alone at 14 days post-injury.These findings indicate that functional 4E-BP1 and S6K1 are neuroprotective and that 4E-BP1 may exert protective effects through a pathway at least partially independent of Rhe b/mTOR.Together,our results show that constitutively active Rheb promotes the survival of retinal ganglion cells and axon regeneration through modulating S6K1 and 4E-BP1 activity.Phosphorylated S6K1 and 4E-BP1 promote axon regeneration but play an antagonistic role in the survival of retinal ganglion cells.

RhoA as a target to promote neuronal survival and axon regeneration

Paralysis following spinal cord injury （SCI） is due to failure of axonal regeneration. It is believed that the capacities of neurons to regrow their axons are due partly to their intrinsic characteristics, which in turn are greatly influenced by several types of inhibitory molecules that are present, or even increased in the extracellular environment of the injured spinal cord. Many of these inhibitory molecules have been studied extensively in recent years. It has been suggested that the small GTPase RhoA is an intracellular convergence point for signaling by these extracellular inhibitory molecules, but due to the complexity of the central nervous system （CNS） in mammals, and the limitation of pharmacological tools, the specific roles of RhoA are unclear. By exploiting the anatomical and technical advantages of the lamprey CNS, we recently demonstrated that RhoA knockdown promotes true axon regeneration through the lesion site after SCI. In addition, we found that RhoA knockdown protects the large, identified reticulospinal neurons from apoptosis after their axons were axotomized in spinal cord. Therefore, manipulation of the RhoA signaling pathway may be an important approach in the development of treatments that are both neuroprotective and axon regeneration-promoting, to enhance functional recovery after SCI.

Brain delivering RNA-based therapeutic strategies by targeting mTOR pathway for axon regeneration after central nervous system injury

Injuries to the central nervous system(CNS)such as stroke,brain,and spinal cord trauma often result in permanent disabilities because adult CNS neurons only exhibit limited axon regeneration.The brain has a surprising intrinsic capability of recovering itself after injury.However,the hostile extrinsic microenvironment significantly hinders axon regeneration.Recent advances have indicated that the inactivation of intrinsic regenerative pathways plays a pivotal role in the failure of most adult CNS neuronal regeneration.Particularly,substantial evidence has convincingly demonstrated that the mechanistic target of rapamycin(mTOR)signaling is one of the most crucial intrinsic regenerative pathways that drive axonal regeneration and sprouting in various CNS injuries.In this review,we will discuss the recent findings and highlight the critical roles of mTOR pathway in axon regeneration in different types of CNS injury.Importantly,we will demonstrate that the reactivation of this regenerative pathway can be achieved by blocking the key mTOR signaling components such as phosphatase and tensin homolog(PTEN).Given that multiple mTOR signaling components are endogenous inhibitory factors of this pathway,we will discuss the promising potential of RNA-based therapeutics which are particularly suitable for this purpose,and the fact that they have attracted substantial attention recently after the success of coronavirus disease 2019 vaccination.To specifically tackle the blood-brain barrier issue,we will review the current technology to deliver these RNA therapeutics into the brain with a focus on nanoparticle technology.We will propose the clinical application of these RNA-mediated therapies in combination with the brain-targeted drug delivery approach against mTOR signaling components as an effective and feasible therapeutic strategy aiming to enhance axonal regeneration for functional recovery after CNS injury.

Identification of key genes involved in axon regeneration and Wallerian degeneration by weighted gene co-expression network analysis

Peripheral nerve injury repair requires a certain degree of cooperation between axon regeneration and Wallerian degeneration.Therefore,investigating how axon regeneration and degeneration work together to repair peripheral nerve injury may uncover the molecular mechanisms and signal cascades underlying peripheral nerve repair and provide potential strategies for improving the low axon regeneration capacity of the central nervous system.In this study,we applied weighted gene co-expression network analysis to identify differentially expressed genes in proximal and distal sciatic nerve segments from rats with sciatic nerve injury.We identified 31 and 15 co-expression modules from the proximal and distal sciatic nerve segments,respectively.Functional enrichment analysis revealed that the differentially expressed genes in proximal modules promoted regeneration,while the differentially expressed genes in distal modules promoted neurodegeneration.Next,we constructed hub gene networks for selected modules and identified a key hub gene,Kif22,which was up-regulated in both nerve segments.In vitro experiments confirmed that Kif22 knockdown inhibited proliferation and migration of Schwann cells by modulating the activity of the extracellular signal-regulated kinase signaling pathway.Collectively,our findings provide a comparative framework of gene modules that are co-expressed in injured proximal and distal sciatic nerve segments,and identify Kif22 as a potential therapeutic target for promoting peripheral nerve injury repair via Schwann cell proliferation and migration.All animal experiments were approved by the Institutional Animal Ethics Committee of Nantong University,China(approval No.S20210322-008)on March 22,2021.

Silencing the enhancer of zeste homologue 2,Ezh2,represses axon regeneration of dorsal root ganglion neurons

Recovery from injury to the peripheral nervous system is different from that of the central nervous system in that it can lead to gene reprogramming that can induce the expression of a series of regeneration-associated genes.This eventually leads to axonal regeneration of injured neurons.Although some regeneration-related genes have been identified,the regulatory network underlying axon regeneration remains largely unknown.To explore the regulator of axon regeneration,we performed RNA sequencing of lumbar L4 and L5 dorsal root ganglion(DRG)neurons at different time points(0,3,6,12 hours,1,3 and 7 days)after rat sciatic nerve crush.The isolation of neurons was carried out by laser capture microscopy combined with NeuN immunofluorescence staining.We found 1228 differentially expressed genes in the injured sciatic nerve tissue.The hub genes within these differentially expressed genes include Atf3,Jun,Myc,Ngf,Fgf2,Ezh2,Gfap and Il6.We verified that the expression of the enhancer of zeste homologue 2 gene(Ezh2)was up-regulated in DRG neurons after injury,and this up-regulation differed between large-and small-sized dorsal root ganglion neurons.To investigate whether the up-regulation of Ezh2 impacts axonal regeneration,we silenced Ezh2 with siRNA in cultured DRG neurons and found that the growth of the newborn axons was repressed.In our investigation into the regulatory network of Ezh2 by interpretive phenomenal analysis,we found some regulators of Ezh2(including Erk,Il6 and Hif1a)and targets(including Atf3,Cdkn1a and Smad1).Our findings suggest that Ezh2,as a nerve regeneration-related gene,participates in the repair of the injured DRG neurons,and knocking down the Ezh2 in vitro inhibits the axonal growth of DRG neurons.All the experimental procedures approved by the Administration Committee of Experimental Animals of Jiangsu Province of China(approval No.S20191201-201)on March 21,2019.

Extrinsic and intrinsic mechanisms of axon regeneration:the need for spinal cord injury treatment strategies to address both

Spinal cord injury（SCI）causes disturbances in motor and sensory functions leading to paralysis,the severity of which depends on the spinal level of the injury.Traumatic lesions of spinal cord axon projection tracts are untreatable in human patients,although numerous research groupsworldwide are studying putative treatment strategies.

Can lithium enhance the extent of axon regeneration and neurological recovery following peripheral nerve trauma?

The clinical"gold standard"technique for attempting to restore function to nerves with a gap is to bridge the gap with sensory autografts.However,autografts induce good to excellent recovery only across short nerve gaps,in young patients,and when repairs are performed a short time post nerve trauma.Even under the best of conditions,<50%of patients recover good recovery.Although many alternative techniques have been tested,none is as effective as autografts.Therefore,alternative techniques are required that increase the percentage of patients who recover function and the extent of their recovery.This paper examines the actions of lithium,and how it appears to trigger all the cellular and molecular events required to promote axon regeneration,and how both in animal models and clinically,lithium administration enhances both the extent of axon regeneration and neurological recovery.The paper proposes more extensive clinical testing of lithium for its ability and reliability to increase the extent of axon regeneration and functional recovery.

Synapsing with NG2 cells(polydendrocytes),unappreciated barrier to axon regeneration?

Have you heard of NG2 cells or NG2 glia or polydendro- cytes~. These are new names for the precursor cells that used to be referred to as oligodendrocyte precursor cells （OPCs）, which become the oligodendrocytes that myelinate central nervous system （CNS） axons. Evidence suggests, however, that they have other functions, besides differentiating into oligodendrocytes. Most notably, the OPCs/NG2 cells are uni- formly distributed in grey matter as well as in white matter, which matches poorly with the distribution of myelinating oligodendrocytes. Furthermore, not every NG2 cell is fated to become an oligodendrocyte. Hence the term OPC can be fairly applied only when discussing the role of these cells in the oligodendrocyte lineage.

Mesenchymal stem cell therapy for retinal ganglion cell neuroprotection and axon regeneration

Retinal ganglion cells （RGCs） are responsible for propagat- ing signals derived from visual stimuli in the eye to the brain, along their axons within the optic nerve to the superior colliculus, lateral geniculate nucleus and visu- al cortex of the brain. Damage to the optic nerve either through trauma, such as head injury, or degenerative dis- ease, such as glaucoma causes irreversible loss of function through degeneration of non-regenerating RGC axons and death of irreplaceable RGCs, ultimately leading to blindness （Berry et al., 2008）. The degeneration of RGCs and their axons is due to the loss of the necessary source of retrogradely transported neurotrophic factors （NTFs） being hindered by axonal injury. NTFs are survival factors for neurons and play a pivotal part in axon regeneration. Stem cells particularly mesenchymal stem cells （MSCs） have been shown to possess a natural intrinsic capacity for paracrine support, releasing multiple signalling mol- ecules including NTFs. By transplanting MSCs into the vitreous, they are positioned adjacent to the injured reti- na to provide paracrine-mediated therapy for the retinal neuronal cells （Johnson et al., 2010a; Mead et al., 2013）. Additionally, MSCs may be pre-differentiated into sup- portive glial-like cells, such as Schwann cells, which could further increase their potential for paracrine support of injured neurons （Martens et al., 2013）. Thus, MSCs have received considerable attention as a new cellular therapy for both traumatic and degenerative eye disease, acting as an alternative source of NTFs, protecting injured RGCs and promoting regeneration of their axons （Figure 1）.

Regeneration-associated macrophages： a novel approach to boost intrinsic regenerative capacity for axon regeneration

Axons in central nervous system （CNS） do not regenerate spontaneously after injuries such as stroke and traumatic spinal cord iniury. Both intrinsic and extrinsic factors are responsible for the regeneration fail- ure, Although intensive research efforts have been invested on extrinsic regeneration inhibitors, the extent to which glial inhibitors contribute to the regeneration failure in viva still remains elusive. Recent exper- imental evidence has rekindled interests in intrinsic factors for the regulation of regeneration capacity in adult mammals. In this review, we propose that activating macrophages with pro-regenerative molecular signatures could be a novel approach for boosting intrinsic regenerative capacity of CNS neurons. Using a conditioning injury model in which regeneration of central branches of dorsal root ganglia sensory neu- rons is enhanced by a preceding injury to the peripheral branches, we have demonstrated that perineuronal macrophages surrounding dorsal root ganglia neurons are critically involved in the maintenance of en- hanced regeneration capacity. Neuron-derived chemokine （C-C motif） ligand 2 （CCL2） seems to mediate neuron-macrophage interactions conveying injury signals to perineuronal macrophages taking on a soley pro-regenerative phenotype, which we designate as regeneration-associated macrophages （RAMs）. Ma- nipulation of the CCL2 signaling could boost regeneration potential mimicking the conditioning injury, suggesting that the chemokine-mediated RAM activation could be utilized as a regenerative therapeutic strategy for CNS injuries.

Promoting axon regeneration in the central nervous system by increasing PI3-kinase signaling

Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system.Axons in the central nervous system fail to regenerate,meaning that injuries or diseases that cause loss of axonal connectivity have life-changing consequences.In 2008,genetic deletion of PTEN was identified as a means of stimulating robust regeneration in the optic nerve.PTEN is a phosphatase that opposes the actions of PI3-kinase,a family of enzymes that function to generate the membrane phospholipid PIP_(3) from PIP_(2)(phosphatidylinositol(3,4,5)-trisphosphate from phosphatidylinositol(4,5)-bisphosphate).Deletion of PTEN therefore allows elevated signaling downstream of PI3-kinase,and was initially demonstrated to promote axon regeneration by signaling through mTOR.More recently,additional mechanisms have been identified that contribute to the neuron-intrinsic control of regenerative ability.This review describes neuronal signaling pathways downstream of PI3-kinase and PIP3,and considers them in relation to both developmental and regenerative axon growth.We briefly discuss the key neuron-intrinsic mechanisms that govern regenerative ability,and describe how these are affected by signaling through PI3-kinase.We highlight the recent finding of a developmental decline in the generation of PIP_(3) as a key reason for regenerative failure,and summarize the studies that target an increase in signaling downstream of PI3-kinase to facilitate regeneration in the adult central nervous system.Finally,we discuss obstacles that remain to be overcome in order to generate a robust strategy for repairing the injured central nervous system through manipulation of PI3-kinase signaling.

Cholesterol synthesis inhibition or depletion in axon regeneration

Cholesterol is biosynthesized by all animal cells. Beyond its metabolic role in steroidogenesis, it is enriched in the plasma membrane where it has key structural and regulatory functions. Cholesterol is thus presumably important for post-injury axon regrowth, and this notion is supported by studies showing that impairment of local cholesterol reutilization impeded regeneration. However, several studies have also shown that statins, inhibitors of 3-hydroxy-3-methylglutaryl-Co A reductase, are enhancers of axon regeneration, presumably acting through an attenuation of the mevalonate isoprenoid pathway and consequent reduction in protein prenylation. Several recent reports have now shown that cholesterol depletion, as well as inhibition of cholesterol synthesis per se, enhances axon regeneration. Here, I discussed these findings and propose some possible underlying mechanisms. The latter would include possible disruptions to axon growth inhibitor signaling by lipid raft-localized receptors, as well as other yet unclear neuronal survival signaling process enhanced by cholesterol lowering or depletion.

The necessary role of mTORC1 in central nervous system axon regeneration

Permanent loss of vital functions after central nervous system （CNS） injury, e.g., blindness in traumatic optic nerve （ON） injury or paralysis in spinal cord injury, occurs in part because axons in the adult mammalian CNS do not regenerate after injury. Growth failure is due to the diminished intrinsic regenerative capacity of mature neurons and the inhibitory environment of the adult CNS. Neutralizing extracellular inhibitory molecules genetically or pharmacologically yields only limited regeneration and functional recovery, highlighting the critical importance of neuron-intrinsic factors.

Targeting cell surface receptors for axon regeneration in the central nervous system

Axon regeneration in the CNS is largely unsuccessful due to excess inhibitory extrinsic factors within lesion sites together with an intrinsic inability of neurons to regrow following injury. Recent work demonstrates that forced expression of certain neuronal transmembrane receptors can recapitulate neuronal growth resulting in successful growth within and through inhibitory lesion environments. More specifically, neuronal expression of integrin receptors such as alpha9beta1 integrin which binds the extracellular matrix glycoprotein tenascin-C, trk receptors such as trk B which binds the neurotrophic factor BDNF, and receptor PTPσ which binds chondroitin sulphate proteoglycans, have all been show to significantly enhance regeneration of injured axons. We discuss how reintroduction of these receptors in damaged neurons facilitates signalling from the internal environment of the cell with the external environment of the lesion milieu, effectively resulting in growth and repair following injury. In summary, we suggest an appropriate balance of intrinsic and extrinsic factors are required to obtain substantial axon regeneration.

Co-culture of oligodendrocytes and neurons can be used to assess drugs for axon regeneration in the central nervous system

We present a novel in vitro model in which to investigate the efficacy of experimental drugs for the promotion of axon regeneration in the central nervous system. We co-cultured rat hippocampal neurons and cerebral cortical oligodendrocytes, and tested the co-culture system using a Nogo-66 receptor antagonist peptide（NEP1–40）, which promotes axonal growth. Primary cultured oligodendrocytes suppressed axonal growth in the rat hippocampus, but NEP1–40 stimulated axonal growth in the co-culture system. Our results confirm the validity of the neuron-oligodendrocyte co-culture system as an assay for the evaluation of drugs for axon regeneration in the central nervous system.

RAFting the rapids of axon regeneration signaling

The functional regeneration of damaged axons and severed connections in the mature central nervous sys- tem （CNS） remains a challenging goal of neurological research. Mature CNS neurons are refractory to axon regeneration for two major reasons, one, because the ac- tivity of cell-intrinsic mechanisms that drive axon growth during development is low- and often further suppressed after an injury - and two, because certain molecules that are part of mature extracellular matrix and myelin act as strong inhibitors of axon growth. Genetic removal of growth inhibitory molecules can increase axon sprouting, but is not sufficient to enable long-range axon growth. Since axon growth is robust during early developmental stages, it has long been hypothesized that mature injured neurons may be ＂reprogrammed＂ to the earlier growth state by re-activation of the intracellular growth signaling cascades that drive axon elongation in the developing fetus.

Casein kinase signaling in axon regeneration

Recent studies suggest that cell cycle pathways may contain therapeutic targets important for neurotrauma.An example of this is the finding that the vertebrate cell cycle exploits proteolysis pathways,yet these activities persist in fully differentiated cells that have exited the cell cycle such as neurons.We have known for some time that a ubiquitin ligase.

Combined treatment promotes the long-range axon regeneration to right brain targets

Axons in the peripheral nervous system(PNS)can regenerate after injury.However,the adult mammalian central nervous system(CNS)loses the intrinsic regrowth ability.No robust axon regeneration occurs spontaneously after nerve injury,which was clearly observed by Ramon y Cajal in the early 20^(th) century(1,2).Due to lack

Decellularized optic nerve functional scaffold transplant facilitates directional axon regeneration and remyelination in the injured white matter of the rat spinal cord

Axon regeneration and remyelination of the damaged region is the most common repair strategy for spinal cord injury.However,achieving good outcome remains difficult.Our previous study showed that porcine decellularized optic nerve better mimics the extracellular matrix of the embryonic porcine optic nerve and promotes the directional growth of dorsal root ganglion neurites.However,it has not been reported whether this material promotes axonal regeneration in vivo.In the present study,a porcine decellularized optic nerve was seeded with neurotrophin-3-overexpressing Schwann cells.This functional scaffold promoted the directional growth and remyelination of regenerating axons.In vitro,the porcine decellularized optic nerve contained many straight,longitudinal channels with a uniform distribution,and microscopic pores were present in the channel wall.The spatial micro topological structure and extracellular matrix were conducive to the adhesion,survival and migration of neural stem cells.The scaffold promoted the directional growth of dorsal root ganglion neurites,and showed strong potential for myelin regeneration.Furthermore,we transplanted the porcine decellularized optic nerve containing neurotrophin-3-overexpressing Schwann cells in a rat model of T10 spinal cord defect in vivo.Four weeks later,the regenerating axons grew straight,the myelin sheath in the injured/transplanted area recovered its structure,and simultaneously,the number of inflammatory cells and the expression of chondroitin sulfate proteoglycans were reduced.Together,these findings suggest that porcine decellularized optic nerve loaded with Schwann cells overexpressing neurotrophin-3 promotes the directional growth of regenerating spinal cord axons as well as myelin regeneration.All procedures involving animals were conducted in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Sun Yat-sen University(approval No.SYSU-IACUC-2019-B034)on February 28,2019.