Axons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses.

Background: Intricate regulation of the growth cone cytoskeleton controls growth cone dynamics. Results: Loss of CRMP4 disrupts growth cone cytoskeletal dynamics, growth cone expansion, and axon growth. Conclusion: CRMP4 regulates both the actin and microtubule growth cone cytoskeleton. Significance: CRMP4 plays a critical role in regulating cytoskeletal dynamics underlying growth cone properties.

Thyroid hormone (T(3)) regulates the growth and differentiation of rat cerebellar astrocytes. Previously, we have demonstrated that these effects are due, at least in part, to the increased expression of extracellular matrix molecules and growth factors, such as fibroblast growth factor-2. T(3) also modulates neuronal development in an astrocyte-mediated manner. In the mammalian central nervous system, excitatory neurotransmission is mediated mainly by glutamate. However, excessive stimulation of glutamate receptors can lead to excitotoxicity and cell death. Astrocytic glutamate transporters, GLT-1 and GLAST, play an essential role in the clearance of the neuronal-released glutamate from the extracellular space and are essential for maintaining physiological extracellular glutamate levels in the brain. In the present study, we showed that T(3) significantly increased glutamate uptake by cerebellar astrocytes compared with control cultures. Inhibitors of glutamate uptake, such as L-PDC and DL-TBOA, abolished glutamate uptake on control or T(3)-treated astrocytes. T(3) treatment of astrocytes increased both mRNA levels and protein expression of GLAST and GLT-1, although no significant changes on the distribution of these transporters were observed. The gliotoxic effect of glutamate on cultured cerebellar astrocytes was abolished by T(3) treatment of astrocytes. In addition, the neuronal viability against glutamate challenge was enhanced on T(3)-treated astrocytes, showing a putative neuroprotective effect of T(3). In conclusion, our results showed that T(3) regulates extracellular glutamate levels by modulating the astrocytic glutamate transporters. This represents an important mechanism mediated by T(3) on the improvement of astrocytic microenvironment in order to promote neuronal development and neuroprotection.

The small GTPase RhoA regulates the actin cytoskeleton to affect multiple cellular processes including endocytosis, migration and adhesion. RhoA activity is tightly regulated through several mechanisms including GDP/GTP cycling, phosphorylation, glycosylation and prenylation. Previous reports have also reported that cleavage of the carboxy-terminus inactivates RhoA. Here, we describe a novel mechanism of RhoA proteolysis that generates a stable amino-terminal RhoA fragment (RhoA-NTF). RhoA-NTF is detectable in healthy cells and tissues and is upregulated following cell stress. Overexpression of either RhoA-NTF or the carboxy-terminal RhoA cleavage fragment (RhoA-CTF) induces the formation of disorganized actin stress fibres. RhoA-CTF also promotes the formation of disorganized actin stress fibres and nuclear actin rods. Both fragments disrupt the organization of actin stress fibres formed by endogenous RhoA. Together, our findings describe a novel RhoA regulatory mechanism.

The receptor deleted in colorectal cancer (DCC) mediates the attraction of growing axons to netrin-1 during brain development. In response to netrin-1 stimulation, DCC becomes a signaling platform to recruit proteins that promote axon outgrowth and guidance. The Ras GTPase-activating protein (GAP) p120RasGAP inhibits Ras activity and mediates neurite retraction and growth cone collapse in response to repulsive guidance cues. Here we show an interaction between p120RasGAP and DCC that positively regulates netrin-1-mediated axon outgrowth and guidance in embryonic cortical neurons. In response to netrin-1, p120RasGAP is recruited to DCC in growth cones and forms a multiprotein complex with focal adhesion kinase and ERK. We found that Ras/ERK activities are elevated aberrantly in p120RasGAP-deficient neurons. Moreover, the expression of p120RasGAP Src homology 2 (SH2)-SH3-SH2 domains, which interact with the C-terminal tail of DCC, is sufficient to restore netrin-1-dependent axon outgrowth in p120RasGAP-deficient neurons. We provide a novel mechanism that exploits the scaffolding properties of the N terminus of p120RasGAP to tightly regulate netrin-1/DCC-dependent axon outgrowth and guidance.

During development, thalamocortical (TC) axons form branches in an activity-dependent fashion. Here we investigated how neuronal activity is converted to molecular signals, focusing on an epigenetic mechanism involving histone deacetylases (HDACs). Immunohistochemistry demonstrated that HDAC9 was translocated from the nucleus to the cytoplasm of thalamic cells during the first postnatal week in rats. In organotypic co-cultures of the thalamus and cortex, fluorescent protein-tagged HDAC9 also exhibited nuclueocytoplasmic translocation in thalamic cells during culturing, which was reversed by tetrodotoxin treatment. Transfection with a mutant HDAC9 that interferes with the translocation markedly decreased TC axon branching in the culture. Similarly, TC axon branching was significantly decreased by the mutant HDAC9 gene transfer in vivo. However, axonal branching was restored by disrupting the interaction between HDAC9 and myocyte-specific enhancer factor 2 (MEF2). Taken together, the present results demonstrate that the nucleocytoplasmic translocation of HDAC9 plays a critical role in activity-dependent TC axon branching by affecting transcriptional regulation and downstream signaling pathways.

Astrocytes clearly play a role in neuronal development. An indirect mechanism of thyroid hormone (T3) in the regulation of neuronal development mediated by astrocytes has been proposed. T3 alters the production and organization of the extracellular matrix (ECM) proteins and proteoglycans, producing a high-quality substrate for neuronal differentiation. The present study investigated the effect of hypothyroidism on the astrocyte production of fibronectin (FN) and laminin (LN) as well as their involvement in neuronal growth and neuritogenesis. Our results demonstrated that the amount of both FN and LN were significantly reduced in cultures of hypothyroid astrocytes from rat cerebellum compared with normal cells. This effect was accompanied by reduced numbers of neurons and neuritogenesis. Similarly, the proportions of neurons and neurons with neurites were reduced in cultures on ECM prepared from hypothyroid astrocytes in comparison with normal cells. The proportion of both normal and hypothyroid neurons is strongly reduced in astrocyte ECM compared with cocultures on astrocyte monolayers, suggesting that extracellular factors other than ECM proteins are involved in this process. Moreover, treatment of hypothyroid astrocytic cultures with T3 restored the area of both FN and LN immunostaining to normal levels and partially reestablished neuronal survival and neuritogenesis. Taken together, our results demonstrated that hypothyroidism involves impairment of the astrocytic microenvironment and affects the production of ECM proteins. Thus, hypothyroidism is implicated in impaired neuronal development.

The identification of molecular processes involved in regulating neurite outgrowth is an active area of interest for investigators studying neural development and regeneration. In vitro assays designed to measure growth cone morphology and neurite length are frequently used to assess neuronal responses to developmental guidance cues and inhibitory cues that exist in the adult CNS. Here, we describe the procedures to assess morphological responses of cultured dorsal root ganglion neurons to attractive and repellent cues, with a focus on repellents found in the injured adult CNS. The chapter describes methods to culture the DRGs, apply inhibitory ligands, and assess morphological responses. These assays provide biological readouts to assess the capacity of a molecule to act as an inhibitory or growth promoting cue. The readouts can be used as screening tools to aid in the identification of novel targets or drugs for promoting nerve regeneration.