Guidance Of Nerve Growth Cones Research Articles

Regulation of chemotropic guidance of nerve growth cones by microRNA

BackgroundThe small non-coding microRNAs play an important role in development by regulating protein translation, but their involvement in axon guidance is unknown. Here, we investigated the role of microRNA-134 (miR-134) in chemotropic guidance of nerve growth cones.ResultsWe found that miR-134 is highly expressed in the neural tube of Xenopus embryos. Fluorescent in situ hybridization also showed that miR-134 is enriched in the growth cones of Xenopus spinal neurons in culture. Importantly, overexpression of miR-134 mimics or antisense inhibitors blocked protein synthesis (PS)-dependent attractive responses of Xenopus growth cones to a gradient of brain-derived neurotrophic factor (BDNF). However, miR-134 mimics or inhibitors had no effect on PS-independent bidirectional responses of Xenopus growth cones to bone morphogenic protein 7 (BMP7). Our data further showed that Xenopus LIM kinase 1 (Xlimk1) mRNA is a potential target of miR-134 regulation.ConclusionsThese findings demonstrate a role for miR-134 in translation-dependent guidance of nerve growth cones. Different guidance cues may act through distinct signaling pathways to elicit PS-dependent and -independent mechanisms to steer growth cones in response to a wide array of spatiotemporal cues during development.

Effects of Ultraviolet-A and Riboflavin on the Interaction of Collagen and Proteoglycans during Corneal Cross-linking

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

Cyclic GMP-Gated CNG Channels Function in Sema3A-Induced Growth Cone Repulsion

Cyclic nucleotide-gated channels (CNGCs) transduce external signals required for sensory processes, e.g., photoreception, olfaction, and taste. Nerve growth cone guidance by diffusible attractive and repulsive molecules is regulated by differential growth cone Ca2+ signaling. However, the Ca2+-conducting ion channels that transduce guidance molecule signals are largely unknown. We show that rod-type CNGC-like channels function in the repulsion of cultured Xenopus spinal neuron growth cones by Sema3A, which triggers the production of the cGMP that activates the Xenopus CNGA1 (xCNGA1) subunit-containing channels in interneurons. Downregulation of xCNGA1 or overexpression of a mutant xCNGA1 incapable of binding cGMP abolished CNG currents and converted growth cone repulsion to attraction in response to Sema3A. We also show that Ca2+ entry through xCNGCs is required to mediate the repulsive Sema3A signal. These studies extend our knowledge of the function of CNGCs by demonstrating their requirement for signal transduction in growth cone guidance.

Sensing the Right Path

During nervous system development, axonal growth cones find their way to their targets with the help of various chemoattractant and chemorepellant cues. Several neurite guidance signals modulate calcium influx into the growth cone, but the underlying mechanisms and the specific channels involved have been unclear (see Gomez). Now two groups, Li et al. and Wang and Poo, provide evidence implicating transient receptor potential canonical (TRPC) channels--a family of channels known for mediating sensory responses--in the response to the chemoattractants brain-derived neurotrophic factor (BDNF) and netrin-1. Wang and Poo performed perforated whole-cell patch clamp analysis of cultured Xenopus spinal neurons and found that netrin-1 elicited membrane depolarization that was inhibited by an antibody to its receptor, DDC. Netrin-induced current present during pharmacological blockade of most voltage-dependent ion channels was inhibited by SKF-96365, which blocks TRP channels, as was a similar current produced by BDNF. Further, growth cone turning in response to netrin-1 or BDNF was abolished by SKF-96365. Injection of morpholino oligonucleotides (MOs) that knocked down xTRPC1 expression inhibited the putative TRP currents and abolished growth cone turning in response to a netrin-1 gradient. Moreover, MO or SKF-96365 abolished the netrin-induced increase in growth cone calcium concentration. Li et al. investigated TRPC channels in BDNF-mediated guidance of cultured cerebellar granule cells, which was blocked by antibodies to the TrkB receptor, incubation with a membrane-permeable calcium chelator, or pharmacological depletion of intracellular calcium stores. Although pharmacological blockade of several voltage-sensitive sodium and calcium channels had no effect on growth cone turning elicited by BDNF, turning was abolished by SKF-96365, as well as by pharmacological inhibition of phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), or blockade of the inositol 1,4,5-trisphosphate receptor (IP 3 R). In contrast, a PLC activator elicited turning, an effect that depended on extracellular calcium and was abolished by SKF-96365. BDNF increased growth cone calcium concentration, an effect that depended on both calcium influx and release from internal stores and that was reduced by SKF-96365, pharmacological inhibition of PLC, or IP 3 R blockade. BDNF-induced turning was abolished by siRNA directed against TRPC3 or by overexpression of dominant-negative mutant forms of TRPC3 or TRPC6. Thus, activation of TRPC channels appears to play a key role in calcium-dependent neurite pathfinding in response to both netrin-1 and BDNF. Y. Li, Y.-C. Jia, K. Cui, N. Li, Z.-Y. Zheng, Y.-Z. Wang, X.-B. Yuan, Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor. Nature 434 , 894-898 (2005). [PubMed] G. X. Wang, M.-M. Poo, Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434 , 898-904 (2005). [PubMed] T. Gomez, Channels for pathfinding. Nature 434 , 835-838 (2005). [PubMed]

Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) is known to promote neuronal survival and differentiation and to guide axon extension both in vitro and in vivo. The BDNF-induced chemo-attraction of axonal growth cones requires Ca2+ signalling, but how Ca2+ is regulated by BDNF at the growth cone remains largely unclear. Extracellular application of BDNF triggers membrane currents resembling those through TRPC (transient receptor potential canonical) channels in rat pontine neurons and in Xenopus spinal neurons. Here, we report that in cultured cerebellar granule cells, TRPC channels contribute to the BDNF-induced elevation of Ca2+ at the growth cone and are required for BDNF-induced chemo-attractive turning. Several members of the TRPC family are highly expressed in these neurons, and both Ca2+ elevation and growth-cone turning induced by BDNF are abolished by pharmacological inhibition of TRPC channels, overexpression of a dominant-negative form of TRPC3 or TRPC6, or downregulation of TRPC3 expression via short interfering RNA. Thus, TRPC channel activity is essential for nerve-growth-cone guidance by BDNF.

GPCRs Mediate Axon Repulsion

Chemokines, first identified for chemoattractant activity for immune cells, are now becoming recognized as migratory signals in the central nervous system. So far, they have been implicated in both neuron and glial cell migration (see the Related Resources). Xiang et al. provide evidence that a gradient of the chemokine stromal-derived factor-1 (SDF-1) can repel axon growth of cultured cerebellar granule cells, promoting repulsive axon turning that was inhibited by an antibody against the chemokine receptor CXCR4. Chemokines activate G protein-coupled receptors (GPCRs). In Xenopus spinal neurons, repulsive turning was stimulated by the addition of a gradient of the metabotropic (GPCR) γ-aminobutyric acid (GABA B ) receptor activator, baclofen. GABA itself promoted chemoattraction of these neurons through stimulation of the ionotropic GABA A receptor. Chemorepulsion was inhibited by inhibitors of G i or phospholipase C (PLC) or depletion of internal calcium stores. Repulsion could be converted to attraction by the addition of the guanosine 3',5'-monophosphate (cGMP) analog 8-Br-cGMP or by the addition of protein kinase C (PKC) inhibitors. The authors suggest that a gradient of GPCR activation can stimulate growth cone turning. Whether the response is an attractive or repulsive one depends on the cGMP concentration. Y. Xiang, Y. Li, Z. Zhang, K. Cui, S. Wang, X.-b. Yuan, C.-p. Wu, M.-m. Poo, S. Duan, Nerve growth cone guidance mediated by G protein-coupled receptors, Nature Neurosci. 5 , 843-8448 (2002). [Online Journal]

Adaptation in the chemotactic guidance of nerve growth cones.

Pathfinding by growing axons in the developing nervous system may be guided by gradients of extracellular guidance factors. Analogous to the process of chemotaxis in microorganisms, we found that axonal growth cones of cultured Xenopus spinal neurons exhibit adaptation during chemotactic migration, undergoing consecutive phases of desensitization and resensitization in the presence of increasing basal concentrations of the guidance factor netrin-1 or brain-derived neurotrophic factor. The desensitization is specific to the guidance factor and is accompanied by a reduction of Ca2+ signalling, whereas resensitization requires activation of mitogen-associated protein kinase and local protein synthesis. Such adaptive behaviour allows the growth cone to re-adjust its sensitivity over a wide range of concentrations of the guidance factor, an essential feature for long-range chemotaxis.

Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance.

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF resulted in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways.

The trkA Receptor Mediates Growth Cone Turning toward a Localized Source of Nerve Growth Factor

We have developed an in vitro system for studying the interaction of chick dorsal root ganglion neuronal growth cones with a localized source of nerve growth factor (NGF) covalently conjugated to polystyrene beads. Growth cones rapidly turned and migrated under NGF-coated beads in a process that involved the initial formation of persistent contact with a bead, followed by directed flow of cytoplasm toward the point of contact. A role for the local activation of the high-affinity NGF receptor trkA was suggested by a strong inhibition of the turning response by (1) the addition of an antibody against the extracellular portion of trkA, (2) the elevation of the background concentration of NGF to saturate trkA, or (3) the presence of a concentration of the drug K252a that inhibits trkA activation. NGF binding to the pan-neurotrophin receptor p75 is also involved but is not required for turning. These data show a new role for both the trkA and the p75 receptors: the mediation of local events in the guidance of nerve growth cones.