The role of RhoA interacting proteins in the Nogo signalling pathway of axon outgrowth inhibition /

Regrowth in the lesioned central nervous system is impeded by inhibitory molecules including myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs). Inhibitory molecules engage neuronal cell surface receptors and activate the small GTPase RhoA in injured neurons to mediate neurite outgrowth inhibition through targeted modifications to the cytoskeleton. Inhibition of RhoA with the ribosyltransferase C3 attenuates neurite outgrowth inhibition in vitro and in vivo but the ubiquitous expression and multifunctionality of RhoA may limit the specificity of therapeutic RhoA antagonists. The hypothesis of the thesis is that molecules that functionally interact with RhoA to mediate myelin-dependent inhibition may represent more specific targets for therapeutic intervention. We have explored the contribution of two RhoA interacting proteins to the neurite outgrowth inhibitory effects of MAIs. In Chapter 2 we describe the contribution of the rho effector, Rho kinase (ROCK) to MAI responses in neurons. In Chapter 3 we identify the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a novel RhoA binding partner that mediates neuronal responses to CNS inhibitors. By structure function analysis we have developed a molecular antagonist of CRMP4b-RhoA binding that promotes neurite outgrowth on inhibitory substrates in vitro and has the potential to be a potent and specific molecular therapeutic for spinal cord injury. In Chapter 4 we identify glycogen sythase kinase 3b (GSK3b) as an important kinase in the MAI pathway that regulates protein interactions with RhoA. This thesis provides insights into the signal transduction machinery that is engaged in response to CNS inhibitors and suggests several novel therapeutic targets to promote axon regeneration following CNS injury

The role of CRMP4 in nerve regenerations

The inability of CNS neurons to regenerate and reform functional connections following spinal cord injury has devastating clinical consequences. The failure of CNS neurons to spontaneously regenerate following injury can be partially attributed to the expression of neurite outgrowth inhibitory myelin associated inhibitors (MAIs). MAIs signal through a tripartite receptor complex to activate the cytosolic protein RhoA and influence cytoskeletal dynamics. RhoA antagonists promote neuronal survival and regeneration in animal models of nerve injury. However, RhoA's potential as a therapeutic target may be limited by its widespread roles in multiples cellular processes and cell types. In an attempt to discover more specific therapeutic targets to promote nerve regeneration, our lab identified the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a protein that functionally interacts with RhoA to mediate neurite outgrowth inhibition. siRNA-mediated knockdown of CRMP4 and blockade of the RhoA-CRMP4b interaction with a competitive peptide (C4RIP) attenuates myelin-dependent neurite outgrowth inhibition. Analysis of the proximal tip of extending axons (growth cones) by time lapse video microscopy reveals that C4RIP regulates filopodial dynamics indicating that C4RIP modulates the actin cytoskeleton. We are currently investigating the in vivo roles of CRMP4 in regeneration in an optic nerve injury model by developing readily deliverable C4RIP and a CRMP4 knockout mouse. Elucidating the role of CRMP4 in nerve regeneration may provide insight into the molecular mechanisms following nervous system injury

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 neuronalinjury. MAIs and CSPGs stimulate intracellular signals including the activation of RhoA and Rho kinase to block axonal extensionthrough targeted modifications to the cytoskeleton. RhoA and ROCK are promising targets for therapeutic intervention to promote CNSrepair; however,their ubiquitous expression will limitthe specificity of drugstargetedtothese molecules.We haveidentifiedthe cytosolicphosphoprotein CRMP4b (collapsin-response mediator protein 4b) as a protein that physically and functionally interacts with RhoA tomediate neurite outgrowth inhibition. Short interfering RNA-mediated knockdown of CRMP4 promotes neurite outgrowth on myelinsubstrates, indicating a critical rolefor CRMP4 in neurite outgrowth inhibition. Disruption of CRMP4b–RhoA binding with a competitiveinhibitor attenuates neurite outgrowth inhibition on myelin and aggrecan substrates. Stimulation of neuronal growth cones with Nogoleads 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 thata RhoA–CRMP4b complex forms in responseto inhibitory challenges inthe growth cone environment and regulates cytoskeletal dynamicsat distinct sites necessary for axon outgrowth inhibition. Competitive inhibition of CRMP4b–RhoA binding suggests a novel, highlyspecific 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