Search

There are no known drugs or drug combinations that promote substantial central nervous system axonal regeneration after injury. We used systems pharmacology approaches to model pathways underlying axonal growth and identify a four-drug combination that regulates multiple subcellular processes in the cell body and axons using the optic nerve crush model in rats. We intravitreally injected agonists HU-210 (cannabinoid receptor-1) and IL-6 (interleukin 6 receptor) to stimulate retinal ganglion cells for axonal growth. We applied, in gel foam at the site of nerve injury, Taxol to stabilize growing microtubules, and activated protein C to clear the debris field since computational models predicted that this drug combination regulating two subcellular processes at the growth cone produces synergistic growth. Physiologically, drug treatment restored or preserved pattern electroretinograms and some of the animals had detectable visual evoked potentials in the brain and behavioral optokinetic responses. Morphology experiments show that the four-drug combination protects axons or promotes axonal regrowth to the optic chiasm and beyond. We conclude that spatially targeted drug treatment is therapeutically relevant and can restore limited functional recovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Siddiq, Johnson, Zorina, Yadaw, Toro, Hansen, Rabinovich, Gregorich, Xiong, Tolentino, Hannila, Kaplan, Blitzer, Filbin, Cardozo, Passaglia and Iyengar.)

Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors, we have detected elevated levels of histone H3 in human CSF 24 h after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor Y-box-binding protein 1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C in vitro , and activated protein C treatment promotes axonal regeneration in the crushed optic nerve in vivo . These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Rap1 is a small GTPase that has been implicated in dendritic development and plasticity. In this study, we investigated the role of Rap1 in axonal growth and its activation in response to neurotrophins and myelin-associated inhibitors. We report that Rap1 is activated by brain-derived neurotrophic factor and that this activation can be blocked by myelin-associated glycoprotein (MAG) or central nervous system myelin, which also induced increases in Rap1GAP1 levels. In addition, we demonstrate that adenoviral overexpression of Rap1 enhances neurite outgrowth in the presence of MAG and myelin, while inhibition of Rap1 activity through overexpression of Rap1GAP1 blocks neurite outgrowth. These findings suggest that Rap1GAP1 negatively regulates neurite outgrowth, making it a potential therapeutic target to promote axonal regeneration., (© 2020 Federation of European Biochemical Societies.)

Remyelination of CNS axons by Schwann cells (SCs) is not efficient, in part due to the poor migration of SCs into the adult CNS. Although it is known that migrating SCs avoid white matter tracts, the molecular mechanisms underlying this exclusion have never been elucidated. We now demonstrate that myelin-associated glycoprotein (MAG), a well known inhibitor of neurite outgrowth, inhibits rat SC migration and induces their death via γ-secretase-dependent regulated intramembrane proteolysis of the p75 neurotrophin receptor (also known as p75 cleavage). Blocking p75 cleavage using inhibitor X (Inh X), a compound that inhibits γ-secretase activity before exposing to MAG or CNS myelin improves SC migration and survival in vitro Furthermore, mouse SCs pretreated with Inh X migrate extensively in the demyelinated mouse spinal cord and remyelinate axons. These results suggest a novel role for MAG/myelin in poor SC-myelin interaction and identify p75 cleavage as a mechanism that can be therapeutically targeted to enhance SC-mediated axon remyelination in the adult CNS. SIGNIFICANCE STATEMENT Numerous studies have used Schwann cells, the myelin-making cells of the peripheral nervous system to remyelinate adult CNS axons. Indeed, these transplanted cells successfully remyelinate axons, but unfortunately they do not migrate far and so remyelinate only a few axons in the vicinity of the transplant site. It is believed that if Schwann cells could be induced to migrate further and survive better, they may represent a valid therapy for remyelination. We show that myelin-associated glycoprotein or CNS myelin, in general, inhibit rodent Schwann cell migration and induce their death via cleavage of the neurotrophin receptor p75. Blockade of p75 cleavage using a specific inhibitor significantly improves migration and survival of the transplanted Schwann cells in vivo ., (Copyright © 2017 the authors 0270-6474/17/375885-15$15.00/0.)

Suboptimal axonal regeneration contributes to the consequences of nervous system trauma and neurodegenerative disease, but the intrinsic mechanisms that regulate axon growth remain unclear. We screened 50,400 small molecules for their ability to promote axon outgrowth on inhibitory substrata. The most potent hits were the statins, which stimulated growth of all mouse- and human-patient-derived neurons tested, both in vitro and in vivo, as did combined inhibition of the protein prenylation enzymes farnesyltransferase (PFT) and geranylgeranyl transferase I (PGGT-1). Compensatory sprouting of motor axons may delay clinical onset of amyotrophic lateral sclerosis (ALS). Accordingly, elevated levels of PGGT1B, which would be predicted to reduce sprouting, were found in motor neurons of early- versus late-onset ALS patients postmortem. The mevalonate-prenylation pathway therefore constitutes an endogenous brake on axonal growth, and its inhibition provides a potential therapeutic approach to accelerate neuronal regeneration in humans., (Copyright © 2016. Published by Elsevier Inc.)

Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG's inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage.

Inhibitory molecules associated with CNS myelin, such as myelin-associated glycoprotein (MAG), represent major obstacles to axonal regeneration following CNS injury. Our laboratory has shown that elevating levels of intracellular cAMP, via application of the nonhydrolyzable analog dibutyryl cAMP (dbcAMP), can block the inhibitory effects of MAG and myelin. We have also shown that elevation of cAMP results in upregulation of arginase I and increased polyamine synthesis. Treatment with putrescine or spermidine blocks myelin-mediated inhibition of neurite outgrowth, but the mechanism underlying this effect has not yet been elucidated. Here we show that cyclin-dependent kinase 5 (Cdk5) is required for dbcAMP and putrescine to overcome MAG-mediated inhibition. The ability of dbcAMP and putrescine to overcome inhibition by MAG is abolished in the presence of roscovitine, a Cdk inhibitor that has greater selectivity for Cdk5, and expression of dominant negative Cdk5 abolishes the ability of dbcAMP or putrescine to enhance neurite outgrowth in the presence of MAG. Importantly, dbcAMP and putrescine increase expression of p35, the neuron-specific activator of Cdk5, and rat DRG neurons transduced with HSV overexpressing p35 can overcome inhibition by MAG. The upregulation of p35 by putrescine is also reflected in increased localization of p35 to neurites and growth cones. Last, we show that putrescine upregulates p35 expression by serving as a substrate for hypusine modification of eIF5A, and that this hypusination is necessary for putrescine's ability to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines may encourage regeneration after CNS injury., (Copyright © 2016 the authors 0270-6474/16/363079-13$15.00/0.)

The adult CNS does not spontaneously regenerate after injury, due in large part to myelin-associated inhibitors such as myelin-associated glycoprotein (MAG), Nogo-A, and oligodendrocyte-myelin glycoprotein. All three inhibitors can interact with either the Nogo receptor complex or paired immunoglobulin-like receptor B. A conditioning lesion of the sciatic nerve allows the central processes of dorsal root ganglion (DRG) neurons to spontaneously regenerate in vivo after a dorsal column lesion. After a conditioning lesion, DRG neurons are no longer inhibited by myelin, and this effect is cyclic AMP (cAMP)- and transcription-dependent. Using a microarray analysis, we identified several genes that are up-regulated both in adult DRGs after a conditioning lesion and in DRG neurons treated with cAMP analogues. One gene that was up-regulated under both conditions is metallothionein (MT)-I. We show here that treatment with two closely related isoforms of MT (MT-I/II) can overcome the inhibitory effects of both myelin and MAG for cortical, hippocampal, and DRG neurons. Intrathecal delivery of MT-I/II to adult DRGs also promotes neurite outgrowth in the presence of MAG. Adult DRGs from MT-I/II-deficient mice extend significantly shorter processes on MAG compared with wild-type DRG neurons, and regeneration of dorsal column axons does not occur after a conditioning lesion in MT-I/II-deficient mice. Furthermore, a single intravitreal injection of MT-I/II after optic nerve crush promotes axonal regeneration. Mechanistically, MT-I/II ability to overcome MAG-mediated inhibition is transcription-dependent, and MT-I/II can block the proteolytic activity of α-secretase and the activation of PKC and Rho in response to soluble MAG., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

We investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) during neurite outgrowth of human embryonic stem cell (hESC)-derived neuronal progenitors. PTEN inhibits phosphoinositide 3-kinase (PI3K)/Akt signaling, a common and central outgrowth and survival pathway downstream of neuronal growth factors. It is known that PTEN inhibition, by either polymorphic mutation or gene deletion, can lead to the development of tumorigenesis (Stambolic et al., ; Tamura et al., ). However, temporary inhibition of PTEN, through pharmacological manipulation, could regulate signaling events such as the PI3K/Akt signaling pathway, leading to enhanced recovery of central nervous system (CNS) injury and disease. We demonstrate that pharmacological inhibition of PTEN in hESC-derived neuronal progenitors significantly increased neurite outgrowth in vitro in a dose- and time-dependent manner. Our results indicate that inhibition of PTEN augments neurite outgrowth beyond that of traditional methods such as elevation of intracellular cyclic adenosine monophosphate (cAMP) levels, and depends on upregulation of the PI3K/Akt signaling pathway and its downstream effectors, such as mammalian target of rapamycin (mTOR). PTEN inhibition also rescued neurite outgrowth over an inhibitory substrate in vitro. These findings indicate a remarkable impact on hESC-derived neuronal progenitor plasticity through PTEN inhibition. Overall, these findings identify a novel therapeutic strategy for neurite outgrowth in CNS injury and disease., (© 2014 Wiley Periodicals, Inc.)

Neurons in the CNS do not regenerate following injury; regeneration is blocked by inhibitory proteins in myelin, such as myelin-associated glycoprotein (MAG). Elevating neuronal levels of the second messenger cAMP overcomes this blocked axonal outgrowth. One way to elevate cAMP is pretreating neurons with neurotrophins, such as brain-derived neurotrophic factor (BDNF). However, pleiotropic effects and poor bioavailability make exogenous administration of neurotrophins in vivo problematic; therefore, alternative targets must be considered. In neurons, two families of adenylyl cyclases synthesize cAMP, transmembrane adenylyl cyclases (tmACs), and soluble adenylyl cyclase (sAC). Here, we demonstrate that sAC is the essential source of cAMP for BDNF to overcome MAG-dependent inhibition of neurite outgrowth. Elevating sAC in rat and mouse neurons is sufficient to induce neurite outgrowth on myelin in vitro and promotes regeneration in vivo. These results suggest that stimulators of sAC might represent a novel therapeutic strategy to promote axonal growth and regeneration., (Copyright © 2014 the authors 0270-6474/14/349281-09$15.00/0.)

Numerous animal model studies in the past decade have demonstrated that pharmacological elevation of cyclic AMP (cAMP) alone, or in combination with other treatments, can promote axonal regeneration after spinal cord injury. Elevation of cAMP via the phosphodiesterase 4 (PDE4) inhibitor, rolipram, decreases neuronal sensitivity to myelin inhibitors, increases growth potential and is neuroprotective. Rolipram's ability to cross the blood-brain barrier makes it a practical and promising treatment for CNS regeneration. However, several studies have questioned the efficacy of rolipram when given alone. The purpose of this investigation was to determine the effects of continuous administration of rolipram, given alone for 2 weeks, following a moderate T10 contusion injury in rat. Functional recovery was evaluated using the 21-point Basso, Beattie and Bresnahan (BBB) locomotor recovery scale and the beam walk. We used three-dimensional (3D) instrumented gait analysis to allow detailed assessment and quantification of hindlimb motion. The amount of the damaged tissue and spared white matter was estimated stereologically. Our results show that administration of rolipram following acute spinal cord contusion results in improved motor performance at each time-point. Dynamic assessment of foot motion during treadmill walking revealed a significantly decreased external rotation during the entire step cycle after 8 weeks in rolipram-treated animals. Stereological analysis revealed no significant differences in lesion volume and length. By contrast, spared white matter was significantly higher in the group treated with rolipram. Our results suggest a therapeutic role for rolipram delivered alone following acute SCI., (Copyright © 2013 Elsevier B.V. All rights reserved.)

After CNS injury, axonal regeneration is limited by myelin-associated inhibitors; however, this can be overcome through elevation of intracellular cyclic AMP (cAMP), as occurs with conditioning lesions of the sciatic nerve. This study reports that expression of secretory leukocyte protease inhibitor (SLPI) is strongly upregulated in response to elevation of cAMP. We also show that SLPI can overcome inhibition by CNS myelin and significantly enhance regeneration of transected retinal ganglion cell axons in rats. Furthermore, regeneration of dorsal column axons does not occur after a conditioning lesion in SLPI null mutant mice, indicating that expression of SLPI is required for the conditioning lesion effect. Mechanistically, we demonstrate that SLPI localizes to the nuclei of neurons, binds to the Smad2 promoter, and reduces levels of Smad2 protein. Adenoviral overexpression of Smad2 also blocked SLPI-induced axonal regeneration. SLPI and Smad2 may therefore represent new targets for therapeutic intervention in CNS injury.

Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG's inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or overexpression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.

Despite advances in promoting axonal regeneration after acute spinal cord injury (SCI), elicitation of bridging axon regeneration after chronic SCI remains a formidable challenge. We report that combinatorial therapies administered 6 weeks, and as long as 15 months, after SCI promote axonal regeneration into and beyond a midcervical lesion site. Provision of peripheral nerve conditioning lesions, grafts of marrow stromal cells, and establishment of NT-3 gradients supports bridging regeneration. Controls receiving partial components of the full combination fail to exhibit bridging. Notably, intraneuronal molecular mechanisms recruited by delayed therapies mirror those of acute injury, including activation of transcriptional activators and regeneration-associated genes. Collectively, these findings provide evidence that regeneration is achievable at unprecedented postinjury time points.

Adult spinal axons do not spontaneously regenerate after injury. However, if the peripheral branch of dorsal root ganglion neurons is lesioned before lesioning the central branch of the same neurons in the dorsal column, these central axons will regenerate and, if cultured, are not inhibited from extending neurites by myelin-associated inhibitors of regeneration such as myelin-associated glycoprotein (MAG). This effect can be mimicked by elevating cAMP and is transcription dependent. The ability of cAMP to overcome inhibition by MAG in culture involves the upregulation of the enzyme arginase I (Arg I) and subsequent increase in synthesis of polyamines such as putrescine. Now we show that a peripheral lesion also induces an increase in Arg I expression and synthesis of polyamines. We also show that the conditioning lesion effect in overcoming inhibition by MAG is initially dependent on ongoing polyamine synthesis but, with time after lesion, becomes independent of ongoing synthesis. However, if synthesis of polyamines is blocked in vivo the early phase of good growth after a conditioning lesion is completely blocked and the later phase of growth, when ongoing polyamine synthesis is not required during culture, is attenuated. We also show that putrescine must be converted to spermidine both in culture and in vivo to overcome inhibition by MAG and that spermidine can promote optic nerve regeneration in vivo. These results suggest that spermidine could be a useful tool in promoting CNS axon regeneration after injury.

Inhibitors of axonal regeneration in myelin are believed to be major contributors to the lack of regeneration in the adult CNS. Three of the four known myelin inhibitors, although very different structurally, interact with the same receptor, NgR. However, the absence of NgR has no effect on inhibition of neurite outgrowth in culture, and there is no improvement in CST regeneration in vivo. In a recent issue of Science, a second receptor for these myelin inhibitors was described, PirB, a receptor first described in the immune system. Will PirB be the answer to CST regeneration in vivo?

The environment of the adult CNS prevents axonal regeneration after injury. This inhibition of axonal regeneration can be blocked by elevating cAMP. Previously, we showed that the cAMP pathway can be activated via pre-treatment with neurotrophins and requires activation of several signaling pathways which converge at activation of the transcription factor, CREB. Here, we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is necessary for the neurotrophin-induced phosphorylation of CREB and the block of myelin-mediated inhibition of axonal growth. Pharmacological inhibition of CaMKIV or over-expression of a dominant-negative mutant form of CaMKIV blocks the neurotrophin effect. Interestingly, CaMKIV activation is not necessary if cAMP levels is already elevated. Finally, calcium flux from intracellular stores is necessary for this CaMKIV signaling. These results demonstrate that CaMKIV is another player in the neurotrophin-induced signaling which leads to axonal regeneration and therefore, is a potential target for therapeutic intervention following injury to the adult CNS.

The failure of axons to regenerate after spinal cord injury remains one of the greatest challenges facing both medicine and neuroscience, but in the last 20 years there have been tremendous advances in the field of spinal cord injury repair. One of the most important of these has been the identification of inhibitory proteins in CNS myelin, and this has led to the development of strategies that will enable axons to overcome myelin inhibition. Elevation of intracellular cyclic AMP (cAMP) has been one of the most successful of these strategies, and in this review we examine how cAMP signaling promotes axonal regeneration in the CNS. Intracellular cAMP levels can be increased through a peripheral conditioning lesion, administration of cAMP analogues, priming with neurotrophins or treatment with the phosphodiesterase inhibitor rolipram, and each of these methods has been shown to overcome myelin inhibition both in vitro and in vivo. It is now known that the effects of cAMP are transcription dependent, and that cAMP-mediated activation of CREB leads to upregulated expression of genes such as arginase I and interleukin-6. The products of these genes have been shown to directly promote axonal regeneration, which raises the possibility that other cAMP-regulated genes could yield additional agents that would be beneficial in the treatment of spinal cord injury. Further study of these genes, in combination with human clinical trials of existing agents such as rolipram, would allow the therapeutic potential of cAMP to be fully realized.

Myelin-associated glycoprotein (MAG) is a potent inhibitor of axonal regeneration. It contains five Ig-like domains and is a sialic binding protein. Previously, we showed that the sialic acid binding site on MAG maps to arginine 118 in Ig domain 1 (Kelm et al., 1994). However, sialic acid binding was neither necessary nor sufficient for MAG to bring about inhibition of neurite outgrowth. Consistent with this, we now map a distinct inhibition site on MAG to Ig domain 5 (Ig-5). We show that when a truncated form of MAG missing Ig domains 1 and 2 is expressed by Chinese hamster ovary (CHO) cells, it does not bind sialic acid, but still inhibits neurite outgrowth almost as effectively as full-length MAG. To determine whether the inhibition site mapped to Ig-3, Ig-4, or Ig-5, we made chimeric molecules of various combinations of these three MAG Ig domains fused to Ig domains from another Ig family member, sialoadhesin (Sn), which also binds to sialic acid in the same linkage as MAG. The MAG-Sn molecules were expressed in CHO cells and all contained five Ig domains and were able to bind sialic acid. However, only the chimeric molecules containing MAG Ig-5 inhibited neurite outgrowth. Furthermore, peptides corresponding to sequences in MAG Ig-5, but not Ig-4 or Sn Ig-5, are able to block inhibition of neurite outgrowth by both wild-type MAG and CNS myelin. We conclude that the inhibition site on MAG is carried by Ig domain 5 and that this site is distinct from the sialic-acid binding site.

Numerous studies in the last two decades have resulted in significant progress in our understanding of the role of inhibitors on axonal regeneration and conditions that influence mature neurons to regrow in an inhibitory environment. These studies have revealed putative therapeutic targets and strategies to interfere in the inhibitory signaling cascade and promote axonal regeneration. Some agents that were successful in animal models are now being tested in human patients. All of these advances have raised hope of a cure for an injury that was once thought to be 'an ailment for which nothing is done' (Quote from Edwin Smith surgical papyrus, 1600BC).

In the past decade there has been an explosion in our understanding, at the molecular level, of why axons in the adult, mammalian central nervous system (CNS) do not spontaneously regenerate while their younger counterparts do. Now a number of inhibitors of axonal regeneration have been described, some of the receptors they interact with to transduce the inhibitory signal are known, as are some of the steps in the signal transduction pathway that is responsible for inhibition. In addition, developmental changes in the environment and in the neurons themselves are also now better understood. This knowledge in turn reveals novel, putative sites for drug development and therapeutic intervention after injury to the brain and spinal cord. The challenge now is to determine which of these putative treatments are the most effective and if they would be better applied in combination rather than alone. In this review I will summarize what we have learnt about these molecules and how they signal. Importantly, I will also describe approaches that have been shown to block inhibitors and encourage regeneration in vivo. I will also speculate on what the differences are between the neonatal and adult CNS that allow the former to regenerate and the latter not to.

Lesioning the peripheral branch of a dorsal root ganglion (DRG) neuron before injury of the central branch of the same neuron enables spontaneous regeneration of these spinal axons. This effect is cAMP and transcription dependent. Here, we show that the cytokine interleukin-6 (IL-6) is upregulated in DRG neurons after either a conditioning lesion or treatment with dibutyryl-cAMP. In culture, IL-6 allows neurons to grow in the presence of inhibitors of regeneration present in myelin. Importantly, intrathecal delivery of IL-6 to DRG neurons blocks inhibition by myelin both in vitro and in vivo, effectively mimicking the conditioning lesion. Blocking IL-6 signaling has no effect on the ability of cAMP to overcome myelin inhibitors. Consistent with this, IL-6-deficient mice respond to a conditioning lesion as effectively as wild-type mice. We conclude that IL-6 can mimic both the cAMP effect and the conditioning lesion effect but is not an essential component of either response.

The three known inhibitors of axonal regeneration present in myelin--MAG, Nogo, and OMgp--all interact with the same receptor complex to effect inhibition via protein kinase C (PKC)-dependent activation of the small GTPase Rho. The transducing component of this receptor complex is the p75 neurotrophin receptor. Here we show that MAG binding to cerebellar neurons induces alpha- and then gamma-secretase proteolytic cleavage of p75, in a protein kinase C-dependent manner, and that this cleavage is necessary for both activation of Rho and inhibition of neurite outgrowth.

The lack of axonal growth after injury in the adult central nervous system (CNS) is due to several factors including the formation of a glial scar, the absence of neurotrophic factors, the presence of growth-inhibitory molecules associated with myelin and the intrinsic growth-state of the neurons. To date, three inhibitors have been identified in myelin: Myelin-Associated Glycoprotein (MAG), Nogo-A, and Oligodendrocyte-Myelin glycoprotein (OMgp). In previous studies we reported that MAG inhibits axonal regeneration by high affinity interaction (K(D) 8 nM) with the Nogo66 receptor (NgR) and activation of a p75 neurotrophin receptor (p75NTR)-mediated signaling pathway. Similar to other axon guidance molecules, MAG is bifunctional. When cultured on MAG-expressing cells, dorsal root ganglia neurons (DRG) older than post-natal day 4 (PND4) extend neurites 50% shorter on average than when cultured on control cells. In contrast, MAG promotes neurite outgrowth from DRG neurons from animals younger than PND4. The response switch, which is also seen in retinal ganglia (RGC) and Raphe nucleus neurons, is concomitant with a developmental decrease in the endogenous neuronal cAMP levels. We report that artificially increasing cAMP levels in older neurons can alter their growth-state and induce axonal growth in the presence of myelin-associated inhibitors.

Inhibitors in myelin play a major role in preventing spontaneous axonal regeneration after CNS injury. Elevation of cAMP overcomes this inhibition, in a transcription-dependent manner, through the upregulation of Arginase I (Arg I) and increased synthesis of polyamines. Here, we show that the cAMP effect requires activation of the transcription factor cAMP response element binding protein (CREB) to overcome myelin inhibitors; a dominant-negative CREB abolishes the effect, and neurons expressing a constitutively active form of CREB are not inhibited. Activation of CREB is also required for cAMP to upregulate Arg I, and the ability of constitutively active CREB to overcome inhibition is blocked by an inhibitor of polyamine synthesis. Finally, expression of constitutively active CREB in DRG neurons is sufficient to promote regeneration of subsequently lesioned dorsal column axons. These results indicate that CREB plays a central role in overcoming myelin inhibitors and so encourages regeneration in vivo.

Previous attempts to promote regeneration after spinal cord injury have succeeded in stimulating axonal growth into or around lesion sites but rarely beyond them. We tested whether a combinatorial approach of stimulating the neuronal cell body with cAMP and the injured axon with neurotrophins would propel axonal growth into and beyond sites of spinal cord injury. A preconditioning stimulus to sensory neuronal cell bodies was delivered by injecting cAMP into the L4 dorsal root ganglion, and a postinjury stimulus to the injured axon was administered by injecting neurotrophin-3 (NT-3) within and beyond a cervical spinal cord lesion site grafted with autologous bone marrow stromal cells. One to 3 months later, long-projecting dorsal-column sensory axons regenerated into and beyond the lesion. Regeneration beyond the lesion did not occur after treatment with cAMP or NT-3 alone. Thus, clear axonal regeneration beyond spinal cord injury sites can be achieved by combinatorial approaches that stimulate both the neuronal soma and the axon, representing a major advance in strategies to enhance spinal cord repair.

Although there is no spontaneous regeneration of mammalian spinal axons after injury, they can be enticed to grow if cAMP is elevated in the neuronal cell bodies before the spinal axons are cut. Prophylactic injection of cAMP, however, is useless as therapy for spinal injuries. We now show that the phosphodiesterase 4 (PDE4) inhibitor rolipram (which readily crosses the blood-brain barrier) overcomes inhibitors of regeneration in myelin in culture and promotes regeneration in vivo. Two weeks after a hemisection lesion at C3/4, with embryonic spinal tissue implanted immediately at the lesion site, a 10-day delivery of rolipram results in considerable axon regrowth into the transplant and a significant improvement in motor function. Surprisingly, in rolipram-treated animals, there was also an attenuation of reactive gliosis. Hence, because rolipram promotes axon regeneration, attenuates the formation of the glial scar, and significantly enhances functional recovery, and because it is effective when delivered s.c., as well as post-injury, it is a strong candidate as a useful therapy subsequent to spinal cord injury.

Central neurons regenerate axons if a permissive environment is provided; after spinal cord injury, however, inhibitory molecules are present that make the local environment nonpermissive. A promising new strategy for inducing neurons to overcome inhibitory signals is to activate cAMP signaling. Here we show that cAMP levels fall in the rostral spinal cord, sensorimotor cortex and brainstem after spinal cord contusion. Inhibition of cAMP hydrolysis by the phosphodiesterase IV inhibitor rolipram prevents this decrease and when combined with Schwann cell grafts promotes significant supraspinal and proprioceptive axon sparing and myelination. Furthermore, combining rolipram with an injection of db-cAMP near the graft not only prevents the drop in cAMP levels but increases them above those in uninjured controls. This further enhances axonal sparing and myelination, promotes growth of serotonergic fibers into and beyond grafts, and significantly improves locomotion. These findings show that cAMP levels are key for protection, growth and myelination of injured CNS axons in vivo and recovery of function.

Injury to the adult mammalian central nervous system (CNS) often results in permanent loss of sensory and motor function. This is due to the failure of injured axons to regenerate. The inhibitory nature of the CNS can be attributed to several factors, including formation of the glial scar, the presence of several molecules, associated with myelin, which inhibit axonal regrowth, and the intrinsic growth state of these neurons. Encouraging regeneration in the adult mammalian CNS therefore will require targeting one or all of these factors following injury. Here we illustrate recent work from our laboratory that identifies some of the signalling components involved in modulation of the intrinsic growth state of adult neurons. When activated, these signalling pathways can induce axonal regeneration in the presence of the myelin-associated inhibitors both in vitro and in vivo.

Inhibitors of regeneration in myelin, such as myelin-associated glycoprotein (MAG), play an important role in preventing regeneration after CNS injury. Elevation of cAMP, either with dibutyryl-cAMP (db-cAMP) or by priming with a variety of neurotrophins, overcomes inhibition by MAG and myelin. However, activation of cAMP is not generally regarded as a signaling pathway for neurotrophins. Here we show that the NGF-like neurotrophins overcome inhibition by MAG by activating tyrosine kinase receptors. We also show that activation of extracellular signal-regulated kinase (Erk) by BDNF is required to overcome inhibition by MAG, and that activated Erk transiently inhibits phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP. Inhibition of PDE4 then allows cAMP to increase and so initiates the pathway to overcome inhibition. Furthermore, we also show that basal levels of Erk activation and basal cAMP levels contribute to the effects of db-cAMP by pushing the combined levels of cAMP above a threshold required to overcome inhibition. Together, these results not only show how NGF-like neurotrophins can elevate cAMP and overcome inhibition but also point to a novel mechanism of cross talk in neurons from the Erk to the cAMP signaling pathways.

The past year has yielded many insights and a few surprises in the field of axonal regeneration. The identification of oligodendrocyte-myelin glycoprotein as an inhibitor of axonal growth, and the discovery that the three major myelin-associated inhibitors of CNS regeneration share the same functional receptor, has launched a new wave of studies that aim to identify the signaling components of these inhibitory pathways. These findings also offer new avenues of research directed toward blocking possible therapeutic targets that inhibit regeneration and toward encouraging axonal regeneration in the CNS after injury.

Elevation of cAMP can overcome myelin inhibitors to encourage regeneration of the CNS. We show that a consequence of elevated cAMP is the synthesis of polyamines, resulting from an up-regulation of Arginase I, a key enzyme in their synthesis. Inhibiting polyamine synthesis blocks the cAMP effect on regeneration. Either over-expression of Arginase I or exogenous polyamines can overcome inhibition by MAG and by myelin in general. While MAG/myelin support the growth of young DRG neurons, they become inhibitory as DRGs mature. Endogenous Arginase I levels are high in young DRGs but drop spontaneously at an age that coincides with the switch from promotion to inhibition by MAG/myelin. Over-expressing Arginase I in maturing DRGs blocks that switch. Arginase I and polyamines are more specific targets than cAMP for intervention to encourage regeneration after CNS injury.

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.

The adult, mammalian CNS does not regenerate after injury largely because of a glial scar and inhibitors of regeneration in myelin. To date, two myelin inhibitors, myelin-associated glycoprotein (MAG) and Nogo, both transmembrane proteins, have been identified. No secreted inhibitors of regeneration have been described. However, a proteolytic fragment of MAG (dMAG), consisting of the entire extracellular domain, is readily released from myelin and is found in vivo. Here, we show, first, that a soluble, chimeric form of MAG (MAG-Fc), when secreted from CHO cells in a collagen gel and hence in the absence of a fixed substrate, inhibits/deflects neurite outgrowth from P6 dorsal root ganglion (DRG) neurons. This inhibition was blocked when a MAG monoclonal antibody was included in the gel and a control chimera sialoadhesin-Fc (Sn-Fc), which, like MAG, binds neurons in a sialic acid-dependent manner but does not inhibit axonal growth, had no effect. Using the same assay system we showed that factors secreted from damaged white matter inhibited/deflected neurite outgrowth. This inhibition was neutralized when a MAG monoclonal antibody was included in the gel and there was no inhibition when white matter from a MAG knockout mouse was used. Factors secreted from damaged white matter from wild-type mice had no effect on neurite outgrowth from E18 DRG neurons. These results show that factors secreted from damaged white matter inhibit axonal regeneration and that the majority of inhibitory activity can be accounted for by dMAG. Thus, released dMAG is likely to play an important role in preventing regeneration, immediately after injury before the glial scar forms., (Copyright 2001 Academic Press.)

Unlike neonatal axons, mammalian adult axons do not regenerate after injury. Likewise, myelin, a major factor in preventing regeneration in the adult, inhibits regeneration from older but not younger neurons. Identification of the molecular events responsible for this developmental loss of regenerative capacity is believed key to devising strategies to encourage regeneration in adults after injury. Here, we report that the endogenous levels of the cyclic nucleotide, cAMP, are dramatically higher in young neurons in which axonal growth is promoted both by myelin in general and by a specific myelin component, myelin-associated glycoprotein (MAG), than in the same types of neurons that, when older, are inhibited by myelin-MAG. Inhibiting a downstream effector of cAMP [protein kinase A (PKA)] prevents myelin-MAG promotion from young neurons, and elevating cAMP blocks myelin-MAG inhibition of neurite outgrowth in older neurons. Importantly, developmental plasticity of spinal tract axons in neonatal rat pups in vivo is dramatically reduced by inhibition of PKA. Thus, the switch from promotion to inhibition by myelin-MAG, which marks the developmental loss of regenerative capacity, is mediated by a developmentally regulated decrease in endogenous neuronal cAMP levels.

In peripheral nerve myelin, the intraperiod line results from compaction of the extracellular space due to homophilic adhesion between extracellular domains (ECD) of the protein zero (P(0)) glycoprotein. Point mutations in this region of P(0) cause human hereditary demyelinating neuropathies such as Charcot-Marie-Tooth. We describe transgenic mice expressing a full-length P(0) modified in the ECD with a myc epitope tag. The presence of the myc sequence caused a dysmyelinating peripheral neuropathy similar to two distinct subtypes of Charcot-Marie-Tooth, with hypomyelination, altered intraperiod lines, and tomacula (thickened myelin). The tagged protein was incorporated into myelin and was associated with the morphological abnormalities. In vivo and in vitro experiments showed that P(0)myc retained partial adhesive function, and suggested that the transgene inhibits P(0)-mediated adhesion in a dominant-negative fashion. These mice suggest new mechanisms underlying both the pathogenesis of P(0) ECD mutants and the normal interactions of P(0) in the myelin sheath.

The extracellular domains of myelin Po protein interact homophilically and hence hold myelin compact at the intraperiod line. The cytoplasmic domain of Po, however, can also affect the interactions of its extracellular sequences. Po is acylated, mostly with palmitic acid, at Cys 153, just at the transmembrane:cytoplasmic domain interface. Here we show that Po mutated at Cys 153 to alanine (C153A), is not acylated and is not adhesive. Like wild-type Po, C153A Po clusters within the membrane and seems to interact with the cytoskeleton. On the other hand, the rate of turnover of C153A Po in transfected Chinese hamster ovary cells is almost 4 times faster than wild-type Po. The increased instability of C153A Po compared to wild-type Po may account for its loss of adhesion., (Copyright 2000 Wiley-Liss, Inc.)

Myelin is a potent inhibitor of axon regeneration, but has been viewed as just one of many factors that prevent regeneration after injury. So it comes as a surprise that immunization against myelin has been found to allow extensive axon regeneration after injury, without apparent autoimmune-induced demyelination.

The lack of axonal regeneration in the adult mammalian CNS is due to both unfavorable environmental glial factors and the intrinsic neuronal state. Inhibitors associated with myelin and the glial scar have been extensively studies and it has been shown that neutralizing at least some of the inhibitors can lead to improved growth. Meanwhile, important advances have also been made towards our understanding of the neuronal intrinsic state, particularly the intracellular levels of cyclic nucleotide, that influence the capacity of mature CNS neurons to initiate and maintain a regrowth response. It is well recognized that successful regeneration may only be achieved by application of a combination of strategies that both block glial inhibitors and enhance the intrinsic neuronal growth capacity., (Copyright 2000 Wiley-Liss, Inc.)

Table 1 summarizes the results obtained from expressing in CHO cells C21A P0 and N93A P0 alone, and each with wild-type P0. As can be seen, each of these mutated proteins reach the cell surface when expressed alone, but neither is adhesive. Finally, when each of these mutated P0 molecules is expressed with the wild-type P0, wild-type P0 is no longer adhesive. Therefore, both C21A Po and N93A P0 each have a dominant negative effect on adhesion of wild-type P0. This approach to address the functional consequences of mutations in P0 can now be used to assess the effects of different mutations associated with CMT1B.