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.

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 GSK3␤ is directly phosphorylated and inactivated by MAIs, consequently regulating protein-protein interactions that are critical for myelin-dependent inhibition. Inhibition of GSK3␤ mimics the neurite outgrowth inhibitory effect of myelin. The inhibitory effects of GSK3␤ inhibitors and myelin are not additive indicating that GSK3␤ is a major effector of MAIs. Consistent with this, overexpression of GSK3␤ attenuates myelin inhibition. MAI-dependent phosphorylation and inactivation of GSK3␤ 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 GSK3␤ inhibitors. We describe the first example of GSK3␤ inactivation in response to inhibitory ligands and link the neurite outgrowth inhibitory effects of GSK3␤ inhibition directly to CRMP4. These findings raise the possibility that GSK3␤ inhibition will not effectively promote long-distance CNS regeneration following trauma such as spinal cord injury.

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Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation ofN-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.