A spatially specified systems pharmacology therapy for axonal recovery after injury.

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.

Extracellular histones, a new class of inhibitory molecules of CNS axonal regeneration.

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.

Myelin-associated glycoprotein inhibits neurite outgrowth through inactivation of the small GTPase Rap1.

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.

Myelin-Associated Glycoprotein Inhibits Schwann Cell Migration and Induces Their Death.

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.

Protein Prenylation Constitutes an Endogenous Brake on Axonal Growth.

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.

Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG.

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.

Cyclic AMP and Polyamines Overcome Inhibition by Myelin-Associated Glycoprotein through eIF5A-Mediated Increases in p35 Expression and Activation of Cdk5.

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.

Metallothionein-I/II Promotes Axonal Regeneration in the Central Nervous System.

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.

Soluble adenylyl cyclase is necessary and sufficient to overcome the block of axonal growth by myelin-associated factors.

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.

PTEN inhibition enhances neurite outgrowth in human embryonic stem cell-derived neuronal progenitor cells.

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.

Secretory leukocyte protease inhibitor reverses inhibition by CNS myelin, promotes regeneration in the optic nerve, and suppresses expression of the transforming growth factor-ß signaling protein Smad2.

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.

Rolipram promotes functional recovery after contusive thoracic spinal cord injury in rats.

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.

A novel role for PTEN in the inhibition of neurite outgrowth by myelin-associated glycoprotein in cortical neurons.

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.

A large-scale chemical screen for regulators of the arginase 1 promoter identifies the soy isoflavone daidzeinas a clinically approved small molecule that can promote neuronal protection or regeneration via a cAMP-independent pathway.

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.

Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury.

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.

Increased synthesis of spermidine as a result of upregulation of arginase I promotes axonal regeneration in culture and in vivo.

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.

PirB, a second receptor for the myelin inhibitors of axonal regeneration Nogo66, MAG, and OMgp: implications for regeneration in vivo.

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?

BDNF activates CaMKIV and PKA in parallel to block MAG-mediated inhibition of neurite outgrowth.

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 role of cyclic AMP signaling in promoting axonal regeneration after spinal cord injury.

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.

The inhibition site on myelin-associated glycoprotein is within Ig-domain 5 and is distinct from the sialic acid binding site.

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.