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.

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.

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.

Targeting axon growth from neuronal transplants along preformed guidance pathways in the adult CNS.

To re-establish neuronal circuits lost after CNS injury, transplanted neurons must be able to extend axons toward their appropriate targets. Such growth is highly restricted within the adult CNS attributable to the expression of inhibitory molecules and general lack of guidance cues to direct axon growth. This environment typically induces random patterns of growth and aberrant innervation, if growth occurs at all. To target the growth of axons from neuronal transplants, we are using viral vectors to create guidance pathways before neuronal transplantation. In this study, we transplanted postnatal rat dorsal root ganglia neurons into the corpus callosum of adult rats. Replication-incompetent adenoviruses encoding growth or guidance factors were injected along the desired pathway 1 week before cell transplantation, allowing time for sufficient protein expression by host glial cells. With expression of nerve growth factor (NGF) and basic fibroblast growth factor, sensory axons were able to grow along the corpus callosum, across the midline, and toward an NGF-expressing target in either the contralateral striatum or cortex: a distance of 7-8 mm including a 90 degree turn from white matter into gray matter. Furthermore, expression of semaphorin 3A slightly dorsal and lateral to the turning point increased the number of axons turning into the striatal target. These results show that judicious expression of neuron-specific chemoattractant and chemorepellant molecules using viral vectors can support and target axon growth from neuronal transplants in the adult CNS.

The cytokine interleukin-6 is sufficient but not necessary to mimic the peripheral conditioning lesion effect on axonal growth.

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.

Myelin-associated inhibitory signaling and strategies to overcome inhibition.

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).

Cell adhesion molecule L1 modulates nerve-growth-factor-induced CGRP-IR fiber sprouting.

Overexpression of nerve growth factor (NGF) using adenoviruses (Adts) after spinal cord injury induces extensive regeneration and sprouting of calcitonin-gene-related peptide immunoreactive (CGRP-IR) fibers, whereas overexpression of cell adhesion molecules (CAMs) has no effect on the normal distribution of these fibers. Interestingly, co-expression of cell adhesion molecule L1 and NGF significantly decreases (p<0.0001) CGRP-IR fiber sprouting within the spinal cord, when compared to NGF alone. Co-expression of cell adhesion molecules NCAM or N-cadherin had no effect on NGF-induced CGRP-IR fiber sprouting. These data demonstrate that reduced sprouting is specific to L1 co-expression and not other cell adhesion molecules. In vitro studies carried out to address potential mechanisms show that neurite outgrowth over astrocytes overexpressing L1 in the presence of NGF is comparable to controls, indicating that other factors present in vivo might be involved in the L1-mediated reduction in sprouting. One potential factor is semaphorin 3A (sema3A), which mediates growth cone collapse of CGRP-positive axons. Recent studies have shown that L1 is important in sema3A receptor signaling for cortical neurons. In our study, co-expression of sema3A indeed reduces neurite outgrowth from DRG neurons by about 40% on L1-expressing astrocytes. Based on these results, we hypothesize that overexpression of L1 potentiates sema3A signaling resulting in reduced sprouting.