Alyson E. Fournier

Plexin-Neuropilin-1 Complexes Form Functional Semaphorin-3A Receptors

Class 1 and 3 semaphorins repulse axons but bind to different cell surface proteins. We find that the two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1 (NP-1), form a stable complex. Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes.

Rho kinase inhibition enhances axonal regeneration in the injured CNS

Myelin-associated inhibitors limit axonal regeneration in the injured brain and spinal cord. A common target of many neurite outgrowth inhibitors is the Rho family of small GTPases. Activation of Rho and a downstream effector of Rho, p160ROCK, inhibits neurite outgrowth. Here, we demonstrate that Rho is directly activated by the myelin-associated inhibitor Nogo-66. Using a binding assay to measure Rho activity, we detected increased levels of GTP Rho in PC12 and dorsal root ganglion (DRG) cell lysates after Nogo-66 stimulation. Rho activity levels were not affected by Amino–Nogo stimulation. Rho inactivation with C3 transferase promotes neurite outgrowth of chick DRG neurons in vitro, but with the delivery method used here, it fails to promote neurite outgrowth after corticospinal tract (CST) lesions in the adult rat. Inhibition of p160ROCK with Y-27632 also promotes neurite outgrowth on myelin-associated inhibitors in vitro. Furthermore, Y-27632 enhances sprouting of CST fibers in vivo and accelerates locomotor recovery after CST lesions in adult rats.

Structure and axon outgrowth inhibitor binding of the Nogo‐66 receptor and related proteins

The myelin‐derived proteins Nogo, MAG and OMgp limit axonal regeneration after injury of the spinal cord and brain. These cell‐surface proteins signal through multi‐subunit neuronal receptors that contain a common ligand‐binding glycosylphosphatidylinositol‐anchored subunit termed the Nogo‐66 receptor (NgR). By deletion analysis, we show that the binding of soluble fragments of Nogo, MAG and NgR to cell‐surface NgR requires the entire leucine‐rich repeat (LRR) region of NgR, but not other portions of the protein. Despite sharing extensive sequence similarity with NgR, two related proteins, NgR2 and NgR3, which we have identified, do not bind Nogo, MAG, OMgp or NgR. To investigate NgR specificity and multi‐ligand binding, we determined the crystal structure of the biologically active ligand‐binding soluble ectodomain of NgR. The molecule is banana shaped with elongation and curvature arising from eight LRRs flanked by an N‐terminal cap and a small C‐terminal subdomain. The NgR structure analysis, as well as a comparison of NgR surface residues not conserved in NgR2 and NgR3, identifies potential protein interaction sites important in the assembly of a functional signaling complex.

Truncated Soluble Nogo Receptor Binds Nogo-66 and Blocks Inhibition of Axon Growth by Myelin

CNS myelin contains axon outgrowth inhibitors, such as Nogo, that restrict regenerative growth after injury. An understanding of the mechanism of Nogo signaling through its receptor (NgR) is critical to developing strategies for overcoming Nogo-mediated inhibition. Here we analyze the function of NgR domains in outgrowth inhibition. Analysis of alkaline phosphatase (AP)-Nogo binding in COS-7 cells reveals that the leucine-rich repeat domain is necessary and sufficient for Nogo binding and NgR multimerization. Viral infection of embryonic day 7 chick retinal ganglion cells with mutated NgR demonstrates that the NgR C-terminal domain is required for inhibitory signaling but not ligand binding. The NgR glycosylphosphatidylinositol domain is not essential for inhibitory signaling but may facilitate Nogo responses. From this analysis, we have developed a soluble, truncated version of the Nogo receptor that antagonizes outgrowth inhibition on both myelin and Nogo substrates. These data suggest that NgR mediates a significant fraction of myelin inhibition of axon outgrowth.

Repulsive factors and axon regeneration in the CNS

During the past year, a major advance in the study of axon regeneration was the molecular cloning of Nogo. The expression of Nogo protein by CNS myelin may be a major factor in the failure of CNS axon regeneration. The effect of disrupting Nogo-dependent axon inhibition can now be studied conclusively. In related work, immunization with a Nogo-containing CNS myelin preparation was shown to promote regeneration and dramatic functional recovery after spinal cord trauma.

Natalizumab Effects on Immune Cell Responses in Multiple Sclerosis

Our objective was to study in vivo biological effects of natalizumab on immune cell phenotype and function in multiple sclerosis (MS) patients.

NeuriteTracer: a novel ImageJ plugin for automated quantification of neurite outgrowth.

In vitro assays to measure neuronal growth are a fundamental tool used by many neurobiologists studying neuronal development and regeneration. The quantification of these assays requires accurate measurements of neurite length and neuronal cell numbers in neuronal cultures. Generally, these measurements are obtained through labor-intensive manual or semi-manual tracing of images. To automate these measurements, we have written NeuriteTracer, a neurite tracing plugin for the freely available image-processing program ImageJ. The plugin analyzes fluorescence microscopy images of neurites and nuclei of dissociated cultured neurons. Given user-defined thresholds, the plugin counts neuronal nuclei, and traces and measures neurite length. We find that NeuriteTracer accurately measures neurite outgrowth from cerebellar, DRG and hippocampal neurons. Values obtained by NeuriteTracer correlate strongly with those obtained by semi-manual tracing with NeuronJ and by using a sophisticated analysis package, MetaXpress. We reveal the utility of NeuriteTracer by demonstrating its ability to detect the neurite outgrowth promoting capacity of the rho kinase inhibitor Y-27632. Our plugin is an attractive alternative to existing tracing tools because it is fully automated and ready for use within a freely accessible imaging program.

Myelin-Associated Inhibitors Regulate Cofilin Phosphorylation and Neuronal Inhibition through LIM Kinase and Slingshot Phosphatase

Myelin-associated inhibitors (MAIs) signal through a tripartate receptor complex on neurons to limit axon regeneration in the CNS. Inhibitory influences ultimately converge on the cytoskeleton to mediate growth cone collapse and neurite outgrowth inhibition. Rho GTPase and its downstream effector Rho kinase are key signaling intermediates in response to MAIs; however, the links between Rho and the actin cytoskeleton have not been fully defined. We found that Nogo-66, a potent inhibitory fragment of Nogo-A, signals through LIM (LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) kinase and Slingshot (SSH) phosphatase to regulate the phosphorylation profile of the actin depolymerization factor cofilin. Blockade of LIMK1 activation and subsequent cofilin phosphorylation circumvents myelin-dependent inhibition in chick dorsal root ganglion neurons, suggesting that phosphorylation and inactivation of cofilin is critical for neuronal inhibitory responses. Subsequent activation of SSH1 phosphatase mediates cofilin dephosphorylation and reactivation. Overexpression of SSH1 does not mimic the neurite outgrowth inhibitory effects of myelin, suggesting an alternative role in MAI inhibition. We speculate that SSH-mediated persistent cofilin activation may be responsible for maintaining an inhibited neuronal phenotype in response to myelin inhibitors.

Identification of Crmp4 as a Convergent Regulator of Axon Outgrowth Inhibition

Myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs) contribute to failed regeneration after neuronal injury. MAIs and CSPGs stimulate intracellular signals including the activation of RhoA and Rho kinase to block axonal extension through targeted modifications to the cytoskeleton. RhoA and ROCK are promising targets for therapeutic intervention to promote CNS repair; however, their ubiquitous expression will limit the specificity of drugs targeted to these molecules. We have identified the cytosolic phosphoprotein CRMP4b (collapsin-response mediator protein 4b) as a protein that physically and functionally interacts with RhoA to mediate neurite outgrowth inhibition. Short interfering RNA-mediated knockdown of CRMP4 promotes neurite outgrowth on myelin substrates, indicating a critical role for CRMP4 in neurite outgrowth inhibition. Disruption of CRMP4b–RhoA binding with a competitive inhibitor attenuates neurite outgrowth inhibition on myelin and aggrecan substrates. Stimulation of neuronal growth cones with Nogo leads to colocalization of CRMP4b and RhoA at discrete regions within the actin-rich central and peripheral domains of the growth cone, indicative of a potential function in cytoskeletal rearrangements during neurite outgrowth inhibition. Together, these data indicate that a RhoA–CRMP4b complex forms in response to inhibitory challenges in the growth cone environment and regulates cytoskeletal dynamics at distinct sites necessary for axon outgrowth inhibition. Competitive inhibition of CRMP4b–RhoA binding suggests a novel, highly specific therapeutic avenue for promoting regeneration after CNS injury.

GSK3β Regulates Myelin-Dependent Axon Outgrowth Inhibition through CRMP4

Myelin-associated inhibitors (MAIs) contribute to failed regeneration in the CNS. The intracellular signaling pathways through which MAIs block axonal repair remain largely unknown. Here, we report that the kinase GSK3β is directly phosphorylated and inactivated by MAIs, consequently regulating protein–protein interactions that are critical for myelin-dependent inhibition. Inhibition of GSK3β mimics the neurite outgrowth inhibitory effect of myelin. The inhibitory effects of GSK3β inhibitors and myelin are not additive indicating that GSK3β is a major effector of MAIs. Consistent with this, overexpression of GSK3β attenuates myelin inhibition. MAI-dependent phosphorylation and inactivation of GSK3β regulate phosphorylation of CRMP4, a cytosolic regulator of myelin inhibition, and its ability to complex with RhoA. Introduction of a CRMP4 antagonist attenuates the neurite outgrowth inhibitory properties of GSK3β inhibitors. We describe the first example of GSK3β inactivation in response to inhibitory ligands and link the neurite outgrowth inhibitory effects of GSK3β inhibition directly to CRMP4. These findings raise the possibility that GSK3β inhibition will not effectively promote long-distance CNS regeneration following trauma such as spinal cord injury.