Gangliosides and Nogo receptors independently mediate myelin-associated glycoprotein inhibition of neurite outgrowth in different nerve cells

In the injured nervous system, myelin-associated glycoprotein (MAG) on residual myelin binds to receptors on axons, inhibits axon outgrowth, and limits functional recovery. Conflicting reports identify gangliosides (GD1a and GT1b) and glycosylphosphatidylinositol-anchored Nogo receptors (NgRs) as exclusive axonal receptors for MAG. We used enzymes and pharmacological agents to distinguish the relative roles of gangliosides and NgRs in MAG-mediated inhibition of neurite outgrowth from three nerve cell types, dorsal root ganglion neurons (DRGNs), cerebellar granule neurons (CGNs), and hippocampal neurons. Primary rat neurons were cultured on control substrata and substrata adsorbed with full-length native MAG extracted from purified myelin. The receptors responsible for MAG inhibition of neurite outgrowth varied with nerve cell type. In DRGNs, most of the MAG inhibition was via NgRs, evidenced by reversal of inhibition by phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves glycosylphosphatidylinositol anchors, or by NEP1-40, a peptide inhibitor of NgR. A smaller percentage of MAG inhibition of DRGN outgrowth was via gangliosides, evidenced by partial reversal by addition of sialidase to cleave GD1a and GT1b or by P4, an inhibitor of ganglioside biosynthesis. Combining either PI-PLC and sialidase or NEP1-40 and P4 was additive. In contrast to DRGNs, in CGNs MAG inhibition was exclusively via gangliosides, whereas inhibition of hippocampal neuron outgrowth was mostly reversed by sialidase or P4 and only modestly reversed by PI-PLC or NEP1-40 in a non-additive fashion. A soluble proteolytic fragment of native MAG, dMAG, also inhibited neurite outgrowth. In DRGNs, dMAG inhibition was exclusively NgR-dependent, whereas in CGNs it was exclusively ganglioside-dependent. An inhibitor of Rho kinase reversed MAG-mediated inhibition in all nerve cells, whereas a peptide inhibitor of the transducer p75(NTR) had cell-specific effects quantitatively similar to NgR blockers. Our data indicate that MAG inhibits axon outgrowth via two independent receptors, gangliosides and NgRs.     

LINGO-1-Fc蛋白对低钾诱导小脑颗粒神经元凋亡的保护作用

髓鞘抑制因子Nogo-A、MAG和OMgp通过共同的受体信号复合物NgR/p75NTR(或者TROY)发挥对中枢神经纤维再生的抑制作用.新近克隆的跨膜蛋白LINGO-1是该信号途径的另一个重要组成成分和调节分子.LINGO-1特异表达于中枢神经系统,神经元上的LINGO-1被证明参与调节中枢神经再生的抑制信号,而少突胶质细胞表达的LINGO-1分子参与负调节少突胶质细胞的髓鞘化过程.为探讨LINGO-1分子在神经元凋亡过程中的作用,利用包含LINGO-1分子胞外段LRR和IgC2结构域的Fc融合蛋白作为功能性拮抗剂,研究LINGO-1对低钾诱导的小脑颗粒神经元凋亡的保护作用.利用成熟的Hoechst标记凋亡细胞的方法,观察到经LINGO-1-Fc蛋白预处理2h能够显著阻止小脑颗粒神经元的凋亡.仅包括LRR结构域的GST-LINGO-1与LINGO-1-Fc蛋白,虽同样具有与颗粒神经元的结合活性,但是GST-LINGO-1不能有效地阻止低钾诱导的细胞凋亡.这些结果提示,LINGO-1-Fc蛋白能够阻止低钾诱导的小脑颗粒神经元凋亡,并且这种作用可能是IgC2结构域依赖的.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Spinal axon regeneration induced by elevation of cyclic AMP

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 p75 receptor transduces the signal from myelin-associated glycoprotein to Rho

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. The receptor for MAG or signals that elicit morphological changes in neurons remained to be established. Here we show that the neurotrophin receptor p75 (p75(NTR)) is the signal transducing element for MAG. Adult dorsal root ganglion neurons or postnatal cerebellar neurons from mice carrying a mutation in the p75(NTR) gene are insensitive to MAG with regard to neurite outgrowth. MAG activates small GTPase RhoA, leading to retarded outgrowth when p75(NTR)) is present. Colocalization of p75(NTR) and MAG binding is seen in neurons. Ganglioside GT1b, which is one of the binding partners of MAG, specifically associates with p75(NTR). Thus, p75(NTR) and GT1b may form a receptor complex for MAG to transmit the inhibitory signals in neurons.     

Molecular dissection of the myelin-associated glycoprotein receptor complex reveals cell type-specific mechanisms for neurite outgrowth inhibition

Neuronal Nogo66 receptor-1 (NgR1) binds the myelin inhibitors NogoA, OMgp, and myelin-associated glycoprotein (MAG) and has been proposed to function as the ligand-binding component of a receptor complex that also includes Lingo-1, p75(NTR), or TROY. In this study, we use Vibrio cholerae neuraminidase (VCN) and mouse genetics to probe the molecular composition of the MAG receptor complex in postnatal retinal ganglion cells (RGCs). We find that VCN treatment is not sufficient to release MAG inhibition of RGCs; however, it does attenuate MAG inhibition of cerebellar granule neurons. Furthermore, the loss of p75(NTR) is not sufficient to release MAG inhibition of RGCs, but p75(NTR-/-) dorsal root ganglion neurons show enhanced growth on MAG compared to wild-type controls. Interestingly, TROY is not a functional substitute for p75(NTR) in RGCs. Finally, NgR1(-/-) RGCs are strongly inhibited by MAG. In the presence of VCN, however, NgR1(-/-) RGCs exhibit enhanced neurite growth. Collectively, our experiments reveal distinct and cell type-specific mechanisms for MAG-elicited growth inhibition.     

A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.     

Differential roles of Rap1 and Rap2 small GTPases in neurite retraction and synapse elimination in hippocampal spiny neurons

The Rap family of small GTPases is implicated in the mechanisms of synaptic plasticity, particularly synaptic depression. Here we studied the role of Rap in neuronal morphogenesis and synaptic transmission in cultured neurons. Constitutively active Rap2 expressed in hippocampal pyramidal neurons caused decreased length and complexity of both axonal and dendritic branches. In addition, Rap2 caused loss of dendritic spines and spiny synapses, and an increase in filopodia-like protrusions and shaft synapses. These Rap2 morphological effects were absent in aspiny interneurons. In contrast, constitutively active Rap1 had no significant effect on axon or dendrite morphology. Dominant-negative Rap mutants increased dendrite length, indicating that endogenous Rap restrains dendritic outgrowth. The amplitude and frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-mediated miniature excitatory postsynaptic currents (mEPSCs) decreased in hippocampal neurons transfected with active Rap1 or Rap2, associated with reduced surface and total levels of AMPA receptor subunit GluR2. Finally, increasing synaptic activity with GABA(A) receptor antagonists counteracted Rap2's inhibitory effect on dendrite growth, and masked the effects of Rap1 and Rap2 on AMPA-mediated mEPSCs. Rap1 and Rap2 thus have overlapping but distinct actions that potentially link the inhibition of synaptic transmission with the retraction of axons and dendrites.     

Myelin-associated glycoprotein protects neurons from excitotoxicity

In addition to supporting rapid nerve conduction, myelination nurtures and stabilizes axons and protects them from acute toxic insults. One myelin molecule that protects and sustains axons is myelin-associated glycoprotein (MAG). MAG is expressed on the innermost wrap of myelin, apposed to the axon surface, where it interacts with axonal receptors that reside in lateral membrane domains including gangliosides, the glycosylphosphatidylinositol-anchored Nogo receptors, and β1-integrin. We report here that MAG protection extends beyond the axon to the neurons from which those axons emanate, protecting them from excitotoxicity. Compared to wild type mice, Mag-null mice displayed markedly increased seizure activity in response to intraperitoneal injection of kainic acid, an excitotoxic glutamate receptor agonist. Mag-null mice also had larger lesion volumes in response to intrastriatal injection of the excitotoxin NMDA. Prior injection of a soluble form of MAG partially protected Mag-null mice from NMDA-induced lesions. Hippocampal neurons plated on proteins extracted from wild-type rat or mouse myelin were resistant to kainic acid-induced excitotoxicity, whereas neurons plated on proteins from Mag-null myelin were not. Protection was reversed by anti-MAG antibody and replicated by addition of soluble MAG. MAG-mediated protection from excitotoxicity was dependent on Nogo receptors and β1-integrin. We conclude that MAG engages membrane-domain resident neuronal receptors to protect neurons from excitotoxicity, and that soluble MAG mitigates excitotoxic damage in vivo.     

Leupeptin enhances cell surface localization of fibroblast growth factor receptor 1 in adult sensory neurons by increased recycling

Fibroblast growth factors (FGFs) act as trophic factors during development and regeneration of the nervous system. FGFs mediate their responses by activation of four types of FGF receptors (FGFR1-4). FGFR1 is expressed in adult sensory neurons of dorsal root ganglia (DRG), and overexpression of FGFR1 enhances FGF-2-induced elongative axon growth in vitro. Ligand-induced activation of FGFR1 is followed by endocytosis and rapid lysosomal degradation. We previously reported that the lysosomal inhibitor leupeptin prevents degradation of FGFR1 and promotes FGF-2-induced elongative axon growth of DRG neurons overexpressing FGFR1. Therefore, we analyzed the effects of leupeptin on intracellular sorting of FGFR1 in PC12 pheochromocytoma cells and DRG neurons. Leupeptin increased colocalization of FGFR1 with lysosomes. Furthermore, leupeptin enhanced the cell surface localization of FGFR1 by increased receptor recycling and this effect was abolished by the recycling inhibitor monensin. In addition, a lysine mutant of FGFR1, which is preferentially recycled back to the cell surface, promoted elongative axon growth of DRG neurons similar to leupeptin. In contrast, the lysosomal inhibitor bafilomycin had no effect on surface localization of FGFR1, inhibited axon growth of DRG neurons and abolished the effects of leupeptin on receptor recycling. Together, our results strongly imply that increased recycling of FGFR1 promotes axon elongation, but not axonal branching, of adult DRG neurons in vitro.     

Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.     

The p75 receptor mediates axon growth inhibition through an association with PIR-B

The Nogo receptor and paired immunoglobulin-like receptor B (PIR-B) are receptors for three myelin-derived axon-growth inhibitors, including myelin-associated glycoprotein (MAG). In this study, we report that the p75 receptor is required for the signal transduction of PIR-B, which interacted with p75 upon ligand binding. In addition, p75 was required for activation of Src homology 2-containing protein tyrosine phosphatase (SHP), which is induced by MAG binding to PIR-B. Mice carrying a mutation in the p75 gene showed promotion of axonal regeneration after optic nerve injury. Thus, our results indicate that p75 has a critical role in axon growth inhibition in specific neuronal tracts.     

TROY Interacts with Rho Guanine Nucleotide Dissociation Inhibitor α (RhoGDIα) to Mediate Nogo-induced Inhibition of Neurite Outgrowth

TROY can functionally substitute p75 to comprise the Nogo receptor complex, which transduces the inhibitory signal of myelin-associated inhibitory factors on axon regeneration following CNS injury. The inhibition of neurite extension relies on TROY-dependent RhoA activation, but how TROY activates RhoA remains unclear. Here, we firstly identified Rho guanine nucleotide dissociation inhibitor α (RhoGDIα) as a binding partner of TROY using GST pull-down combined with two-dimensional gel electrophoresis and mass spectra analysis. The interaction was further confirmed by coimmunoprecipitation in vitro and in vivo. Deletion mutagenesis revealed that two regions of the TROY intracellular domain (amino acids 234–256 and 321–350) were essential for the interaction with RhoGDIα. Secondly, TROY and RhoGDIα were coexpressed in postnatal dorsal root ganglion neurons, cortex neurons, and cerebellar granule neurons (CGNs). Thirdly, TROY/RhoGDIα association was potentiated by Nogo-66 and was independent of p75/RhoGDIα interaction. Fourthly, TROY/RhoGDIα interaction was still able to activate RhoA when p75 was deficient. Furthermore, RhoA activation was decreased dramatically when TROY was knocked down in p75-deficient CGNs cells. Finally, RhoGDIα overexpression abolished RhoA activation and following neurite outgrowth inhibition by Nogo-66 in both wild-type and p75-deficient CGNs. These results showed that the association of RhoGDIα with TROY contributed to TROY-dependent RhoA activation and neurite outgrowth inhibition after Nogo-66 stimulation.     

Binding of soluble myelin-associated glycoprotein to specific gangliosides induces the association of p75NTR to lipid rafts and signal transduction

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. Here we show that gangliosides, GT1b and GD1a, as well as the Nogo receptor, are functional binding partners for soluble MAG-Fc. Postnatal cerebellar neurons from mice deficient in the GalNAcT gene are insensitive to MAG with regard to neurite outgrowth and lack in the activation of RhoA. MAG-Fc or the antibody to GT1b and GD1a elicits recruitment of p75(NTR.) to lipid rafts, specialized microdomain for signal transduction. Disruption of lipid rafts results in abolishment of inhibitory effects of MAG-Fc and the Nogo peptide. These findings establish gangliosides as functional binding partners for soluble MAG. Gangliosides may play a role in translocation of p75(NTR.) to lipid rafts for initiation of the signal transduction.     

Dose and age-dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target-derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF-dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax-/- mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA-positive, and TrkA-/- TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose- and age-dependent fashion, mediated by p75NTR signaling through TrkA expressing axons.     

Getting RIP'd Stunts Your Growth

The p75 neurotrophin receptor (p75NTR) collaborates with the Nogo receptor (NgR) and LINGO-1 to activate RhoA in response to myelin-based growth inhibitors such as myelin-associated glycoprotein (MAG). In this issue of Neuron, Domeniconi et al., in a surprising turn, show that MAG induces intramembrane proteolysis (RIP) of p75NTR and find that p75NTR cleavage is required for MAG-induced RhoA activation and growth inhibition.     

Uptake of lipoproteins for axonal growth of sympathetic neurons

Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration.     

中枢神经再生抑制因子及免疫治疗研究

成体哺乳动物中枢神经损伤后早期轴突再生失败的一个主要原因是由于髓磷脂抑制分子的存在。Nogo、髓磷脂相关糖蛋白以及少突胶质细胞髓磷脂糖蛋白等神经再生抑制因子的发现,大大促进了中枢神经再生分子机制的研究。它们均能独立通过Nogo-66受体产生对轴突再生的抑制效应,髓磷脂抑制分子及其信号转导机制的研究日益成为中枢神经再生的研究热点,髓磷脂及其信号转导分子特别是Nogo-66受体、p75神经营养素受体成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制上述抑制因子及相关受体NgR或p75NTR可能有助于中枢神经损伤的修复,围绕这些抑制因子及其相关受体介导的信号转导途径,人们提出了多种治疗中枢神经损伤的新思路,其中免疫学方法尤其受到关注。     

Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling

Regulation of axon growth is a critical event in neuronal development. Nerve growth factor (NGF) is a strong inducer of axon growth and survival in the dorsal root ganglia (DRG). Paradoxically, high concentrations of NGF are present in the target region where axon growth must slow down for axons to accurately identify their correct targets. Semaphorin3A (Sema3A), a powerful axonal repellent molecule for DRG neurons, is also situated in their target regions. NGF is a modulator of Sema3A-induced repulsion and death. We show that Sema3A is a regulator of NGF-induced neurite outgrowth via the TrkA receptor, independent of its growth cone repulsion activity. First, neurite outgrowth of DRG neurons is more sensitive to Sema3A than repulsion. Second, at concentrations sufficient to significantly inhibit Sema3A-induced repulsion, NGF has no effect on Sema3A-induced axon growth inhibition. Third, Sema3A-induced outgrowth inhibition, but not repulsion activity, is dependent on NGF stimulation. Fourth, Sema3A attenuates TrkA-mediated growth signaling, but not survival signaling, and over-expression of constitutively active TrkA blocks Sema3A-induced axon growth inhibition, suggesting that Sema3A activity is mediated via regulation of NGF/TrkA-induced growth. Finally, quantitative analysis of axon growth in vivo supports the possibility that Sema3A affects axon growth, in addition to its well-documented role in axon guidance. We suggest a model whereby NGF at high concentrations in the target region is important for survival, attraction and inhibition of Sema3A-induced repulsion, while Sema3A inhibits its growth-promoting activity. The combined and cross-modulatory effects of these two signaling molecules ensure the accuracy of the final stages in axon targeting.