Attempting to clone genes involved in nerve growth factor (NGF)-induced neurite extension, we cultured dorsal root ganglion (DRG) explants with either NGF or NT3. Using subtraction methods, cDNAs expressed exclusively in NGF-treated explants were enriched, cloned and sequenced. We identified several known genes, and we also found more than 100 novel gene fragments. One of these, designated AN-1, has stretches of significant homology to the GTPase-activating protein RhoGAP. RhoGAP participates in the control of the Rho family small G-proteins, which have been shown to regulate the actin cytoskeleton as well as neurite outgrowth. A search of EST databases and RT-PCR analysis suggest that at least 13 further homologues of AN-1 exist. We found that immunoprecipitation of AN-1 co-precipitates active RhoA, but not RhoG, Rac or Cdc42. Deletion of the RhoGAP domain in AN-1 eliminates this binding. In situ hybridization shows AN-1 mRNA selectively and highly expressed in embryonic DRG and sympathetic neurons as wel...

Evidence suggests that fibroblast growth factors (FGFs) stimulate axon extension of Xenopus retinal ganglion cell (RGC) neurons. Growth cones express the FGF receptor (FGFR); cells along the optic pathway express FGF-2; FGF-2 promotes axon extension in vitro, and; misexpression of a dominant negative FGFR decreases neurite extension both in vivo and in vitro. The present study aims to determine if FGFs simply promote axon extension or if they chemotropically direct RGC axon trajectories. Thus, we asked if FGF expression alongside the extending optic projection misdirects RGC axons. To determine this, we cotransfected plasmids containing the green fluorescent protein (GFP) and embryonic FGF (eFGF) cDNAs into the neuroepithelium of stage 24 Xenopus embryos. At stage 40, when RGC axons normally have reached the optic tectum, we labeled the axons with horse radish peroxidase (HRP). HRP labeled RGC axons that contact GFP misexpressing neurons along the optic pathway project normally to the tectum. In contrast, ...

The cell adhesion molecule L1 plays an important role in sensory axon pathfinding in the developing chick hindlimb. We have previously shown that when function-blocking anti-L1 antibodies are injected into the limb at St. 25, shortly before most sensory axons enter the plexus, the segmental pattern of sensory projectons is altered. To elucidate how anti-L1 causes pathfinding errors, here we anterogradely labeled small numbers of sensory neurons 5-6 hours after anti-L1 injection and later traced the trajectories of individual axons. We overlaid camera lucida drawings of the plexus with a grid consisting of equally spaced parallel lines, oriented so that it was aligned with the predominant direction of axon growth. We counted how often sensory axons crossed the grid lines and expressed the total number of crossings relative to the number of axons in the region of interest. In anti-L1-injected limbs, the incidence of crossing was nearly twice that of normal embryos. In addition, the frequency of axons turning...

It is widely believed that axons in the central nervous system of adult mammals do not regrow following injury. This failure is thought, at least in part, to underlie the limited recovery of function following injury to the brain or spinal cord. Some studies of fixed tissue have suggested that, counter to dogma, norepinephrine (NE) axons regrow following brain injury. Here, we have used in vivo two-photon microscopy in layer 1 of the primary somatosensory cortex in transgenic mice harboring a fluorophore selectively expressed in NE neurons. This protocol allowed us to explore the dynamic nature of NE axons following injury with the selective NE axon toxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). Following DSP4, NE axons were massively depleted and then slowly and partially recovered their density over a period of weeks. This regrowth was dominated by new axons entering the imaged volume. There was almost no contribution from local sprouting from spared NE axons. Regrown axons did not appear to ...

In the rat frontal cortex GABAergic interneurons are divided into several classes based on the intrinsic firing pattern: fast-spiking (FS) cells, late-spiking (LS) cells and other types (non-FS cells). To reveal morphometric characteristics of cortical interneurons, we reconstructed axonal arborizations of physiologically identified interneuron subtypes in the frontal cortex by Neurolucida, and measured morphological parameters for axonal arbors such as total length, number of branches, mean branch length, number of orders and number of boutons. FS cells showed larger total length and more frequent branching than other types. By Sholl analysis, we quantified the spatial distribution of axonal arbors. Axonal arbors of FS and LS cells distributed more densely near the soma. Non-FS cells with descending axonal arbors were less dense in arborization near the soma and extended the axon collaterals farther. The number of boutons per unit axon length was almost same in all cell types (about 0.2 bouton/µm). By pri...

Axon degeneration is an evolutionarily conserved pathway that eliminates damaged or unneeded axons. Manipulation of this poorly understood pathway may allow treatment of a wide range of neurological disorders. In an RNAi-based screen performed in cultured mouse DRG neurons, we observed strong suppression of injury-induced axon degeneration upon knockdown of Sarm1 [SARM (sterile α-motif-containing and armadillo-motif containing protein)]. We find that a SARM-dependent degeneration program is engaged by disparate neuronal insults: SARM ablation blocks axon degeneration induced by axotomy or vincristine treatment, while SARM acts in parallel with a soma-derived caspase-dependent pathway following trophic withdrawal. SARM is a multidomain protein that associates with neuronal mitochondria. Deletion of the N-terminal mitochondrial localization sequence disrupts SARM mitochondrial localization in neurons but does not alter its ability to promote axon degeneration. In contrast, mutation of either the SAM (sterile...

In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neur...

Growing axons in the CNS often migrate along specific pathways to reach their targets. During embryonic development, this migration is guided by different types of cell adhesion molecules (CAMs) present on the surface of glial cells or other neurons, including the neural cadherin (NCAD). Axons in the adult CNS can be stimulated to regenerate, and travel long distances. Crucially, however, while a few axons are guided effectively through the injured nerve under certain conditions, most axons never migrate properly. The molecular underpinnings of the variable growth, and the glial CAMs that are responsible for CNS axon regeneration remain unclear. Here we used optic nerve crush to demonstrate that NCAD plays multifaceted functions in facilitating CNS axon regeneration. Astrocyte-specific deletion of NCAD dramatically decreases regeneration induced by phosphatase and tensin homolog (PTEN) ablation in retinal ganglion cells (RGCs). Consistent with NCAD’s tendency to act as homodimers, deletion of NCAD in RGCs ...

Cell adhesion is critical for the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule-like extracellular domains that may transduce signals in response to adhesion. By sequence comparison of the extracellular and cytoplasmic domains of the known RPTPs with the DNA database of the Drosophila genome project, we found several loci which may code for new RPTPs. Screening of embryonic cDNA libraries revealed clones encoding two proteins whose genes are closely linked at position 52F. One of these, RPTP52F is a novel receptor tyrosine phosphatase (881 amino acids), that has extracellular domains consisting of multiple fibronectin type III repeats. We have not yet been able to determine the expression pattern for RPTP52F by in situ hybridization. Recently, dsRNA was found to be a potent and specific inhibitor of gene activity in drosophila melanogaster (Kennerdell and Carthew, 1998; Misquitta and Paterson, 1999). Therefore, dsR...

During development, neural cells migrate, extend their axons, arborize and establish appropriate connections with their target cells. Extensive axon arbor remodeling (branch extension and retraction) presumably occurs in conjunction with synapse formation and elimination. We have previously shown that BDNF plays a relevant role during axon arborization, but the question whether the acquired axon arbor complexity correlates with synapse formation remained unresolved. In order to examine the role of BNDF during synapse formation in vivo, we have fluorescently labeled individual Xenopus retinal axons to visualize their morphology while simultaneously expressed a fluorescent chimeric synaptic vesicle protein to selectively visualize sites of synaptic contact at a single cell level. A mixture of two plasmids encoding the Red Fuorescence Protein (RFP) and the fusion protein GFP-Synaptobrevin (Wang and Poo, Soc. Neurosc. Abstr., 1996; Cohen-Cory et al., Soc. Neurosc. Abstr., 1999) were lipofected into the eye of ...