Axon regeneration is hindered by a decline of intrinsic axon growth capability in mature neurons. Reversing this decline is associated with the induction of a large repertoire of regeneration-associated genes (RAGs), but the underlying regulatory mechanisms of the transcriptional changes are largely unknown. Here, we establish a correlation between diminished axon growth potential and histone 4 (H4) hypoacetylation. When neurons are triggered into a growth state, as in the conditioning lesion paradigm, H4 acetylation is restored, and RAG transcription is initiated. We have identified a set of target genes of Smad1, a proregenerative transcription factor, in conditioned DRG neurons. We also show that, during the epigenetic reprogramming process, histone-modifying enzymes work together with Smad1 to facilitate transcriptional regulation of RAGs. Importantly, targeted pharmacological modulation of the activity of histone-modifying enzymes, such as histone deacetylases, leads to induction of multiple RAGs and ...

In a transgenic RNAi based genome-wide screen for RhoGAPs that regulate neuronal morphogenesis (Billuart et al., Cell 107, 195-207, 2001), we found that a knock-down of RacGAP50C activity results in abnormal axon extension and presence of large cells. Detailed phenotypic analysis of MB neurons expressing UAS-RNAi against RacGAP50C revealed its function in regulating axon guidance and in limiting axon branch formation. We have determined that RacGAP50C corresponds to the tumbleweed (tum) gene previously identified based on its mutant phenotypes in the morphogenesis of sensory neuron dendrites (Gao et al., Genes Dev. 13, 2549-61, 1999), and that the tum¹ allele contains a stop codon in RacGAP50C before the GAP domain. MARCM MB clones homozygous for tum¹ exhibits phenotypes remarkably similar to RhoGAP50C RNAi phenotypes. RacGAP50C and its C.elegans and mammalian homologs have previously been shown to regulate cytokinesis in conjunction with its binding partner, a kinesin superfamily member encoded by the pav...

A major challenge for recovery after spinal cord injury is inducing the regeneration of severed spinal axons long after the injury. Zebrafish offer a good model for testing molecules that might induce regeneration of lesioned axons because it is possible to image individually identifiable spinal axons in the intact animal at various times after a lesion and after treatments designed to overcome the injury. Previous studies have shown that severed axons of the Mauthner cell that fail to regenerate immediately after axotomy are still unable to do so weeks after lesioning. Our aim was to test whether cyclic AMP, which has been shown to enhance regrowth when applied prior to injury, could induce Mauthner axons that failed to regenerate to initiate growth and regenerate across the lesion site. We first electroporated fluorescent dye into individual Mauthner cells in order to monitor their regeneration with confocal microscopy. The spinal cord was then lesioned with a large bore glass electrode and the stumps of...

Neurons in the adult mammalian CNS decrease in intrinsic axon growth capacity during development in concert with changes in Krüppel-like transcription factors (KLFs). KLFs regulate axon growth in CNS neurons including retinal ganglion cells (RGCs). Here, we found that knock-down of KLF9, an axon growth suppressor that is normally upregulated 250-fold in RGC development, promotes long-distance optic nerve regeneration in adult rats of both sexes. We identified a novel binding partner, MAPK10/JNK3 kinase, and found that JNK3 (c-Jun N-terminal kinase 3) is critical for KLF9's axon-growth-suppressive activity. Interfering with a JNK3-binding domain or mutating two newly discovered serine phosphorylation acceptor sites, Ser106 and Ser110, effectively abolished KLF9's neurite growth suppression in vitro and promoted axon regeneration in vivo . These findings demonstrate a novel, physiologic role for the interaction of KLF9 and JNK3 in regenerative failure in the optic nerve and suggest new therapeutic strategies...

The secreted semaphorin 3A (Sema3A) belongs to a large protein family that is involved in the patterning of neuronal connections in the developing nervous system of both vertebrates and invertebrates. Sema3A acts as a chemorepellant for many types of axons by activating a receptor complex that contains neuropilin-1 (Nrp-1) as the ligand binding and an A-type plexin as the signal-transducing subunit. In addition to its repulsive effects, Sema3A also acts as an attractant for cortical dendrites. To elucidate the molecular mechanisms underlying Sema3A-mediated chemoattraction, we used PC12 cells as a model system for neuronal differentiation. Sema3A is as effective as NGF in stimulating the extension of neurites by PC12 cells and this response depends on Nrp-1. Inhibition of cGMP production or MAPK activity completely block extension of neurites in response to Sema3A but not to NGF. The repulsive effects of Sema3A are mediated in part by regulating the balance of Rho and Rac activity. Therefore, we analyze th...

The formation of a functional gonadotropin-releasing hormone (GnRH) system requires the successful targeting of GnRH neuron axons to the median eminence (ME). Two distinct processes, general axon outgrowth and the directional extension of the axons, are presumably required for successful targeting. We hypothesize that fibroblast growth factor (FGF) signaling is involved in one or both of these processes. Primary GnRH neurons expressing only green fluorescent protein (GnRH-GFP) and GnRH neurons expressing both GFP and a dominant negative FGF receptor to dampen their FGF responsiveness (GnRH-dnFGFR/GFP) were isolated from two lines of transgenic mice. Each neuronal type was co-cultured with either ME explant or cerebellar (CB) explant for 24 hours. The ability of GnRH-GFP neuron to extend and target neurites to ME and CB was compared with GnRH-dnFGFR/GFP neurons. GnRH-dnFGFR/GFP neurons have significantly shorter neurites compared to GnRH-GFP neurons under all culture conditions. However, both GnRH-GFP and G...

The neural cell adhesion molecule L1 is well known to promote axon growth when coated as a substrate, though L1-L1 homophilic interactions. To investigate how L1 mediates neurite outgrowth, we have transfected cerebellar neurons from L1 knockout mice with various human L1 vectors with mutations or truncations in the L1 cytoplasmic domain and plated these transfected neurons on an L1 substrate. The longest neurite length, branching number, and total neurite length were quantified and compared between the wild-type L1 and mutants. All mutations we tested have remarkably little impact on neurite length. But several mutations affect branching significantly. Removing the alternatively spliced exon RSLE reduces branching by 30%. Removing 110/114 of the amino acids in the L1 cytoplasmic domain reduces the branching by 50% but the length of the longest neurite is largely unaffected. This truncation is likely to disrupt branching due to the loss of L1-ERM binding because the juxtamembrane region of L1 cytoplasmic d...

cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in vivo...

The axon initial segment (AIS) is the site of spike initiation in neurons. Previous studies revealed that spatial distribution of the AIS varies greatly among neurons to meet their specific needs. However, when and how this differentiation arises is unknown. Neurons in the avian nucleus laminaris (NL) are binaural coincidence detectors for sound localization and show differentiation in the distribution of the AIS, with shorter length and a more distal position from the soma with an increase in tuning frequency. We studied these characteristics of the AIS in NL neurons of the chicken during development and found that the AIS differentiates in its distribution after initial formation, and this is driven by activity-dependent and activity-independent mechanisms that differentially regulate distal and proximal boundaries of the AIS. Before hearing onset, the ankyrinG-positive AIS existed at a wide stretch of proximal axon regardless of tuning frequency, but Na+ channels were only partially distributed within t...

The only topographic guidance molecules described to date are EphA receptor tyrosine kinases and their ephrin-A ligands, which control, in part, mapping of the temporal-nasal retinal axis along the anterior-posterior axis of the superior colliculus (SC) through a repellent mechanism. We report here that EphB receptors and ephrin-B ligands direct unique axonal behaviors required for mapping the dorsal-ventral (DV) retinal axis along the lateral-medial (LM) SC axis. EphB2, EphB3, and EphB4 are expressed in a low to high DV gradient in retina and ephrin-B1 and ephrin-B3 are expressed at high levels medially and low levels laterally in the SC. At an age when the retinotopic map resembles its mature form, mice lacking EphB2 and EphB3 exhibit consistent defects in LM topographic mapping. Developmental analyses of wild type mice reveal that retinal axons directionally extend branches along the LM axis to correct their position and establish ordered arborizations. This directed branch extension is disrupted in Eph...