Neurite formation by neurons derived from adult rat hippocampal progenitor cells is susceptible to myelin inhibition

Myelin-associated inhibitors expressed following injury to the adult central nervous system (CNS) induce growth cone collapse and retraction of the axonal cytoskeleton. Myelin-associated glycoprotein (MAG) is a bi-functional molecule that promotes neuritogenesis in some immature neurons during development then becomes inhibitory to neurite outgrowth as neurons mature. Progress is being made towards the elucidation of the downstream events that regulate myelin inhibition of regeneration in neuronal populations. However it is not known how adult-derived neural stem cells or progenitors respond to myelin during neuronal differentiation and neuritogenesis. Here we examine the effect of MAG on neurons derived from an adult rat hippocampal progenitor cell line (AHPCs). We show that, unlike their developmental counterparts, AHPC-derived neurons are susceptible to MAG inhibition of neuritogenesis during differentiation and display a 57% reduction in neurite outgrowth when compared with controls. We demonstrate that this effect can be overcome (by up to 69%) by activation of the neurotrophin, cyclic AMP and protein kinase A pathways or by Rho-kinase suppression. We also demonstrate that combination of these factors enhanced neurite outgrowth from differentiating neurons in the presence of MAG. This work provides important information for the successful generation of new neurons from adult neural stem cell populations within compromised adult circuitry and is thus directly relevant to endogenous repair and regeneration of the adult CNS.     

A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.     

A Novel Microfluidic Device-Based Neurite Outgrowth Inhibition Assay Reveals the Neurite Outgrowth-Promoting Activity of Tropomyosin Tpm3.1 in Hippocampal Neurons

Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system.     

Phagocytic Removal of Neuronal Debris by Olfactory Ensheathing Cells Enhances Neuronal Survival and Neurite Outgrowth via p38MAPK Activity

Compelling evidence from animal models and clinical studies suggest that transplantation of olfactory ensheathing cells (OECs), specialized glia in the olfactory system, combined with specific training may be therapeutically useful in the central nervous system (CNS) injuries and neurodegenerative diseases. The unique function of OECs could mainly attribute to both production of cell adhesion molecules and secretion of growth factors in OECs, which support neuron survival and neurite outgrowth. However, little is known about whether engulfment of neuronal degenerative debris by OECs also equally contributes to neuronal survival and neurite outgrowth. Furthermore, the molecular mechanisms responsible for neuronal degenerative corpses' removal remain elusive. Here, we used an in vitro model of primary culture of spinal cord neurons to investigate the effect of engulfment of degenerative neuron debris by OECs on neuronal survival and neurite outgrowth and the possible molecular mechanisms. Our results showed that OECs can engulf an amount of degenerated neuron debris, and this phagocytosis can make a substantial contribution to neuron growth, as demonstrated by increased number of neurons with longer neurite length and richer neurite branches when compared with the combination of neuron debris and OEC conditioned medium (OECCM). Moreover, p38 mitogen-activated protein kinase (p38MAPK) signaling pathway may mediate the OEC engulfment of debris because the p38MAPK-specific inhibitor, SB203580, can abrogate all the positive effects of OECs, including clearance of degenerated neuron debris and generation of bioactive molecules, indicating that p38MAPK is required for the process of phagocytosis of the neuron debris. In addition, the OEC phagocytic activity had no influence on its generation of bioactive molecules. Therefore, these findings provide new insight into further investigations on the OEC role in the repair of traumatic CNS injury and neurodegenerative diseases.     

Direct Rho-associated kinase inhibiton induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells

Axons fail to regenerate in the adult central nervous system (CNS) following injury. Developing strategies to promote axonal regeneration is therapeutically attractive for various CNS pathologies such as traumatic brain injury, stroke and Alzheimer’s disease. Because the RhoA pathway is involved in neurite outgrowth, Rho-associated kinases (ROCKs), downstream effectors of GTP-bound Rho, are potentially important targets for axonal repair strategies in CNS injuries. We investigated the effects and downstream mechanisms of ROCK inhibition in promoting neurite outgrowth in a PC-12 cell model. Robust neurite outgrowth (NOG) was induced by ROCK inhibitors Y-27632 and H-1152 in a time-and dose-dependent manner. Dramatic cytoskeletal reorganization was noticed upon ROCK inhibition. NOG initiated within 5 to 30 minutes followed by neurite extension between 6 and 10 hours. Neurite processes were then sustained for over 24 hours. Rapid cofilin dephosphorylation was observed within 5 minutes of Y-27632 and H-1152 treatment. Re-phosphorylation was observed by 6 hours after Y-27632 treatment, while H-1152 treatment produced sustained cofilin dephosphorylation for over 24 hours. The results suggest that ROCK-mediated dephosphorylation of cofilin plays a role in the initiation of NOG in PC-12 cells.     

Kinetic and thermodynamic properties of MAG antagonists

Paraplegia is caused by injuries of the central nervous system (CNS) and especially young people suffer from these severe consequences as, for example, the loss of motor functions. The lack of repair of the injured nerve strands originates from the inhibitory environment for axon regeneration in the CNS. Specific inhibitory proteins block the regrowth of nerve roots. One of these neurite outgrowth inhibitors is the myelin-associated glycoprotein (MAG), which is a member of the Siglec family (sialic acid-binding immunoglobulin-like lectin). In previous studies, we identified potent small molecule MAG antagonists. In this communication, we report new neuraminic acid derivatives modified in the 4- and 5-position, and the influence of various structural modifications on their kinetic and thermodynamic binding properties.     

Proteasome inhibition induces neurite outgrowth through posttranslational modification of TrkA receptor

The ubiquitin-proteasome pathway regulates many biological processes, including protein degradation, receptor endocytosis, protein sorting, subnuclear trafficking and neuronal differentiation. While proteasome inhibition is known to induce neurite outgrowth, the signaling mechanisms that mediate these effects have not been defined. In this study, we investigated the underlying mechanisms that link proteasome inhibition with neurite generation. We found that the proteasome inhibitors, MG132 and lactacystin, induced neurite outgrowth and also activated extracellular signal-regulated kinase/mitogen activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways. These proteasome inhibitors also induced phosphorylation and ubiquitination of TrkA receptors, indicating that proteasome inhibition activates the major pathways of TrkA signaling. However, in contrast to nerve growth factor stimulation, which induces internalization of surface TrkA receptors, proteasome inhibitor-induced neurite outgrowth did not require TrkA receptor internalization. These results indicate that the ubiquitin-proteasome system regulates neurite formation through posttranslational modification of TrkA receptors.     

The neuronal differentiation microenvironment is essential for spinal cord injury repair

Spinal cord injury (SCI) often leads to substantial disability due to loss of motor function and sensation below the lesion. Neural stem cells (NSCs) are a promising strategy for SCI repair. However, NSCs rarely differentiate into neurons; they mostly differentiate into astrocytes because of the adverse microenvironment present after SCI. We have shown that myelin-associated inhibitors (MAIs) inhibited neuronal differentiation of NSCs. Given that MAIs activate epidermal growth factor receptor (EGFR) signaling, we used a collagen scaffold-tethered anti-EGFR antibody to attenuate the inhibitory effects of MAIs and create a neuronal differentiation microenvironment for SCI repair. The collagen scaffold modified with anti-EGFR antibody prevented the inhibition of NSC neuronal differentiation by myelin. After transplantation into completely transected SCI animals, the scaffold-linked antibodies induced production of nascent neurons from endogenous and transplanted NSCs, which rebuilt the neuronal relay by forming connections with each other or host neurons to transmit electrophysiological signals and promote functional recovery. Thus, a scaffold-based strategy for rebuilding the neuronal differentiation microenvironment could be useful for SCI repair.     

N-cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces

Receptor-mediated interactions between neurons and astroglia are likely to play a crucial role in the growth and guidance of CNS axons. Using antibodies to neuronal cell surface proteins, we identified two receptor systems mediating neurite outgrowth on cultured astrocytes. N-cadherin, a Ca2(+)-dependent cell adhesion molecule, functions prominently in the outgrowth of neurites on astrocytes by E8 and E14 chick ciliary ganglion (CG) neurons. beta 1-class integrin ECM receptor heterodimers function less prominently in E8 and not at all in E14 neurite outgrowth on astrocytes. The lack of effect of integrin beta 1 antibodies on E14 neurite outgrowth reflects an apparent loss of integrin function, as assayed by E14 neuronal attachment and process outgrowth on laminin. N-CAM appeared not to be required for neurite outgrowth by either E8 or E14 neurons. Since N-cadherin and integrin beta 1 antibodies together virtually eliminated E8 CG neurite outgrowth on cultured astrocytes, these two neuronal receptors are probably important in regulating axon growth on astroglia in vivo.     

A Hypothesis About the Relationship of Myelin-Associated Glycoprotein’s Function in Myelinated Axons to its Capacity to Inhibit Neurite Outgrowth

The myelin-associated glycoprotein (MAG) is selectively localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths suggesting that it functions in glia–axon interactions in the PNS and CNS, and this is supported by much experimental evidence. In addition, MAG is now well known as one of several white matter inhibitors of neurite outgrowth in vitro and axonal regeneration in vivo, and this latter area of research has provided a substantial amount of information about neuronal receptors or receptor complexes for MAG. This article makes the hypothesis that the capacity of MAG to inhibit outgrowth of immature developing or regenerating neurites is an aberration of its normal physiological function to promote the maturation, maintenance, and survival of myelinated axons. The overview summarizes the literature on the function of MAG in PNS and CNS myelin sheaths and its role as an inhibitor of neurite outgrowth to put this hypothesis into perspective. Additional research is needed to determine if receptors and signaling systems similar to those responsible for MAG inhibition of neurite outgrowth also promote the maturation, maintenance, and survival of myelinated axons as hypothesized here, or if substantially different MAG-mediated signaling mechanisms are operative at the glia–axon junction. Special issue article in honor of Dr. George DeVries.     

MARK2 Rescues Nogo-66-Induced Inhibition of Neurite Outgrowth via Regulating Microtubule-Associated Proteins in Neurons In Vitro

The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.     

Maturation of astrocytes in vitro alters the extent and molecular basis of neurite outgrowth

In the developing mammalian central nervous system astrocytes have been proposed as an important substrate for axon growth. In the adult central nervous system following injury, astrocytes are a major component of the gliotic response which has been proposed to block axon growth. Experimental transplantation studies using cultured astrocytes have suggested that immature but not mature cultured astrocytes have the capacity to support axon outgrowth when transplanted into the adult rodent CNS. These observations suggest that astrocyte maturation is accompanied by changes in the functional capacity of these cells to support axon outgrowth. To determine whether this functional change reflects an intrisic astrocyte property, the extent and molecular bases of neurite outgrowth from embryonic rat cortical and chick retinal neurons on cultures of purified immature and mature astrocytes have been compared in vitro. The rate and extent of neurite outgrowth from both neuronal populations are consistently greater over the surface of immature than over the surface of mature astrocytes. Furthermore, antibodies to NCAM and G4/L1 significantly reduce neurite outgrowth on immature but not mature astrocytes, while antibodies to the integrin B1 receptor reduced outgrowth on both immature and, to a lesser extent, mature astrocytes. These results suggest that in vitro mature astrocytes have a reduced capacity and different molecular bases for supporting neurite outgrowth compared to immature astrocytes and are consistent with the proposal that functional changes during astrocyte maturation may partially contribute to regulating axon growth in the mammalian CNS.     

Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite- promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent- extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.     

Nogo-receptors NgR1 and NgR2 do not mediate regulation of CD4 T helper responses and CNS repair in experimental autoimmune encephalomyelitis

Myelin-associated inhibition of axonal regrowth after injury is considered one important factor that contributes to regeneration failure in the adult central nervous system (CNS). Blocking strategies targeting this pathway have been successfully applied in several nerve injury models, including experimental autoimmune encephalomyelitis (EAE), suggesting myelin-associated inhibitors (MAIs) and functionally related molecules as targets to enhance regeneration in multiple sclerosis. NgR1 and NgR2 were identified as interaction partners for the myelin proteins Nogo-A, MAG and OMgp and are probably mediating their growth-inhibitory effects on axons, although the in vivo relevance of this pathway is currently under debate. Recently, alternative functions of MAIs and NgRs in the regulation of immune cell migration and T cell differentiation have been described. Whether and to what extent NgR1 and NgR2 are contributing to Nogo and MAG-related inhibition of neuroregeneration or immunomodulation during EAE is currently unknown. Here we show that genetic deletion of both receptors does not promote functional recovery during EAE and that NgR1 and NgR2-mediated signals play a minor role in the development of CNS inflammation. Induction of EAE in Ngr1/2-double mutant mice resulted in indifferent disease course and tissue damage when compared to WT controls. Further, the development of encephalitogenic CD4(+) Th1 and Th17 responses was unchanged. However, we observed a slightly increased leukocyte infiltration into the CNS in the absence of NgR1 and NgR2, indicating that NgRs might be involved in the regulation of immune cell migration in the CNS. Our study demonstrates the urgent need for a more detailed knowledge on the multifunctional roles of ligands and receptors involved in CNS regeneration failure.     

Recombinant DNA vaccine encoding multiple domains related to inhibition of neurite outgrowth: a potential strategy for axonal regeneration

Myelin-derived proteins, such as tenascin-R (TN-R), myelin associate glycoprotein (MAG), and Nogo-A, inhibit the CNS regeneration. By targeting specifically the inhibitory epitopes, we have investigated whether vaccination with a recombinant DNA molecule encoding multiple domains of myelin inhibitors may be useful in CNS repair. We show here that the recombinant DNA vaccine is able to activate the immune system but does not induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Importantly, it promotes axonal regeneration in a spinal cord injury model. Thus, the application of DNA vaccine, encoding multiple specific domains of major inhibitory proteins and/or their receptors, provides another promising approach to overcome the inhibitory barriers during CNS regeneration.     

Characterization of new cell permeable C3-like proteins that inactivate Rho and stimulate neurite outgrowth on inhibitory substrates

The activation state of Rho is an important determinant of axon growth and regeneration in neurons. Axons can extend neurites on growth inhibitory substrates when Rho is inactivated by C3-ADP-ribosyltransferase (C3). We found by Rho-GTP pull-down assay that inhibitory substrates activate Rho. To inactivate Rho, scrape-loading of C3 was necessary because it does not freely enter cells. To overcome the poor permeability of C3, we made and characterized five new recombinant C3-like chimeric proteins designed to cross the cell membrane by receptor-independent mechanisms. These proteins were constructed by the addition of short transport peptides to the carboxyl-terminal of C3 and tested using a bioassay measuring neurite outgrowth of PC-12 cells plated on growth inhibitory substrates. All five constructs stimulated neurite outgrowth but with different dose-response profiles. Biochemical properties of the chimeric proteins were examined using C3-05, the most effective construct tested. Gel shift assays showed that C3-05 retained the ability to ADP-ribosylate Rho. Western blots and immunocytochemistry were used to verify the presence of C3 inside treated cells. C3-05 was also effective at promoting neurite outgrowth in primary neuronal cultures, as well as causing the disassembly of actin stress fibers and focal adhesions complexes in fibroblasts. These studies demonstrate that the new C3-like proteins are effective in delivering biologically active C3 into different cell types, thereby, inactivating Rho.     

Central Nervous System Regeneration Inhibitors and their Intracellular Substrates

Injury to the central nervous system (CNS) initiates a cascade of responses that is inhibitory to the regeneration of neurons and full recovery. At the site of injury, glial cells conspire with an inhibitory biochemical milieu to construct both physical and chemical barriers that prevent the outgrowth of axons to or beyond the lesion site. These inhibitors include factors derived from myelin, repulsive guidance cues, and chondroitin sulfate proteoglycans. Each bind receptors on the axon surface to initiating intracellular signaling cascades that ultimately result in cytoskeletal reorganization and growth cone collapse. Here, we present an overview of the molecules, receptors, and signaling pathways that inhibit CNS regeneration, with a particular focus on the intracellular signaling machinery that may function as convergent targets for multiple inhibitory ligands.