Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility

Neurotrophin regulation of actin-dependent changes in growth cone motility may depend on the signaling of beta-actin mRNA transport. Formation of an RNP complex between the beta-actin mRNA zipcode sequence and Zipcode Binding Protein 1 (ZBP1) was required for its localization to growth cones. Antisense oligonucleotides to the zipcode inhibited formation of this RNP complex in vitro and the neurotrophin-induced localization of beta-actin mRNA and ZBP1 granules. Live cell imaging of neurons transfected with EGFP-ZBP1 revealed fast, bidirectional movements of granules in neurites that were inhibited by antisense treatment, as visualized by FRAP analysis. NT-3 stimulation of beta-actin protein localization was dependent on the 3'UTR and inhibited by antisense treatment. Growth cones exhibited impaired motility in the presense of antisense. These results suggest a novel mechanism to influence growth cone dynamics involving the regulated transport of mRNA.     

A predominantly nuclear protein affecting cytoplasmic localization of beta-actin mRNA in fibroblasts and neurons.

The localization of beta-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3' untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate pre-mRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of beta-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of beta-actin mRNA.     

The insulin-like growth factor mRNA binding-protein IMP-1 and the Ras-regulatory protein G3BP associate with tau mRNA and HuD protein in differentiated P19 neuronal cells

Tau mRNA is axonally localized mRNA that is found in developing neurons and targeted by an axonal localization signal (ALS) that is located in the 3'UTR of the message. The tau mRNA is trafficked in an RNA-protein complex (RNP) from the neuronal cell body to the distal parts of the axon, reaching as far as the growth cone. This movement is microtubule-dependent and is observed as granules that contain tau mRNA and additional proteins. A major protein contained in the granule is HuD, an Elav protein family member, which has an identified mRNA binding site on the tau 3'UTR and stabilizes the tau message and several axonally targeted mRNAs. Using GST-HuD fusion protein as bait, we have identified four proteins contained within the tau RNP, in differentiated P19 neuronal cells. In this work, we studied two of the identified proteins, i.e. IGF-II mRNA binding protein 1 (IMP-1), the orthologue of chick beta-actin binding protein-ZBP1, and RAS-GAP SH3 domain binding protein (G3BP). We show that IMP-1 associates with HuD and G3BP-1 proteins in an RNA-dependent manner and binds directly to tau mRNA. We also show an RNA-dependent association between G3BP-1 and HuD proteins. These associations are investigated in relation to the neuronal differentiation of P19 cells.     

Long-range signaling in growing neurons after local elevation of cyclic AMP-dependent activity

Cyclic AMP-dependent activity at the growth cone or the soma of cultured Xenopus spinal neurons was elevated by local extracellular perfusion of the neuron with culture medium containing 8-bromoadenosine 3',5'-cyclic monophosphate (8-br-cAMP) or forskolin. During local perfusion of one of the growth cones of multipolar neurons with these drugs, the perfused growth cone showed further extension, while the distant, unperfused growth cones were inhibited in their growth. Local perfusion of the growth cone with culture medium or local perfusion with 8-br-cAMP at a cell-free region 100 microns away from the growth cone did not produce any effect on the extension of the growth cone. Reduced extension of all growth cones was observed when the perfusion with 8-br-cAMP was restricted to the soma. The distant inhibitory effect does not depend on the growth of the perfused growth cone since local coperfusion of the growth cone with 8-br-cAMP and colchicine inhibited growth on both perfused and unperfused growth cones, while local perfusion with colchicine alone inhibited only the perfused growth cone. The distant inhibitory effect was abolished when the perfusion of 8-br-cAMP was carried out together with kinase inhibitor H- 8, suggesting the involvement of cAMP-dependent protein kinase and/or its downstream factors in the long-range inhibitory signaling. Uniform exposure of the entire neuron to bath-applied 8-br-cAMP, however, led to enhanced growth activity at all growth cones. Thus, local elevation of cAMP-dependent activity produces long-range and opposite effects on distant parts of the neuron, and a cytosolic gradient of second messengers may produce effects distinctly different from those following uniform global elevation of the messenger, leading to differential growth regulation at different regions of the same neuron.     

Control of growth cone motility and neurite outgrowth by SPIN90

SPIN90 is an F-actin binding protein thought to play important roles in regulating cytoskeletal dynamics. It is known that SPIN90 is expressed during the early stages of neuronal development, but details of its localization and function in growth cones have not been fully investigated. Our immunocytochemical data show that SPIN90 is enriched throughout growth cones and neuronal shafts in young hippocampal neurons. We also found that its localization correlates with and depends upon the presence of F-actin. Detailed observation of primary cultures of hippocampal neurons revealed that SPIN90 knockout reduces both growth cone areas and in the numbers of filopodia, as compared to wild-type neurons. In addition, total neurite length, the combined lengths of the longest (axonal) and shorter (dendritic) neurites, was smaller in SPIN90 knockout neurons than wild-type neurons. Finally, Cdc42 activity was down-regulated in SPIN90 knockout neurons. Taken together, our findings suggest that SPIN90 plays critical roles in controlling growth cone dynamics and neurite outgrowth.     

Critical role of collapsin response mediator protein-associated molecule CRAM for filopodia and growth cone development in neurons

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. We have previously identified a unique member of this gene family, CRMP-associated molecule CRAM (CRMP-5), which is phylogenetically divergent from the other four CRMPs. In this study, we have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAM in the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. We propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.     

Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.     

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

BDNF Reduces eEF2 Phosphorylation and Enhances Novel Protein Synthesis in the Growth Cones of Dorsal Root Ganglia Neurons

The local translation, which is regulated by extracellular stimuli such as guidance molecules, in growth cones of neurons provides a molecular mechanism for axonal development. In this study, we performed immunocytochemistry together with atomic force microscopy to investigate the localization of ribosomal proteins in the growth cones of rat dorsal root ganglion (DRG) neurons. The immunoreactivity of ribosomal protein P0/1/2 and S6, and novel protein synthesis were observed in the central, sterically bulky region of growth cones. Brain derived neurotrophic factor (BDNF) reduced the eEF2 phosphorylation, indicating its activation, and enhanced protein synthesis within 30 min. The effects of BDNF were completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). These results indicated that BDNF rapidly activates translation and enhances novel protein synthesis in growth cones of DRG though the mTOR signaling.     

Autonomous regulation of growth cone filopodia

The fan-shaped array of filopodia is the first site of contact of a neuronal growth cone with molecules encountered during neuronal pathfinding. Filopodia are highly dynamic structures, and the “action radius” of a growth cone is strongly determined by the length and number of its filopodia. Since interactions of filopodia with instructive cues in the vicinity of the growth cone can have effects on growth cone morphology within minutes, it has to be assumed that a large part of the signaling underlying such morphological changes resides locally within the growth cone proper. In this study, we tested the hypothesis that two important growth cone parameters namely, the length and number of its filopodiaare regulated autonomously in the growth cone. We previously demonstrated in identified neurons from the snail Helisoma trivolvis that filopodial length and number are regulated by intracellular calcium. Here, we investigated filopodial dynamics and their regulation by the second-messenger calcium in growth cones which were physically isolated from their parent neuron by neurite transection. Our results show that isolated growth cones have longer but fewer filopodia than growth cones attached to their parent cell. These isolated growth cones, however, are fully capable of undergoing calcium-induced cytoskeletal changes, suggesting that the machinery necessary to perform changes in filopodial length and number is fully intrinsic to the growth cone proper. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 179–192, 1998     

Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.

Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a neuromodulin-beta-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of neuromodulin and beta-galactosidase is not. A mutant neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target beta-galactosidase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of neuromodulin, may be required for growth cone targeting.     

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.     

Suppression of Radixin and Moesin Alters Growth Cone Morphology,Motility, and Process Formation In Primary Cultured Neurons

In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin–radixin or ezrin–moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin–moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8–10 times slower than the one of growth cones from control neurons. Radixin–moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity.     

Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans.

During nervous system development, neurons form synaptic contacts with distant target cells. These connections are formed by the extension of axonal processes along predetermined pathways. Axon outgrowth is directed by growth cones located at the tips of these neuronal processes. Although the behavior of growth cones has been well-characterized in vitro, it is difficult to observe growth cones in vivo. We have observed motor neuron growth cones migrating in living Caenorhabditis elegans larvae using time-lapse confocal microscopy. Specifically, we observed the VD motor neurons extend axons from the ventral to dorsal nerve cord during the L2 stage. The growth cones of these neurons are round and migrate rapidly across the epidermis if they are unobstructed. When they contact axons of the lateral nerve fascicles, growth cones stall and spread out along the fascicle to form anvil-shaped structures. After pausing for a few minutes, they extend lamellipodia beyond the fascicle and resume migration toward the dorsal nerve cord. Growth cones stall again when they contact the body wall muscles. These muscles are tightly attached to the epidermis by narrowly spaced circumferential attachment structures. Stalled growth cones extend fingers dorsally between these hypodermal attachment structures. When a single finger has projected through the body wall muscle quadrant, the growth cone located on the ventral side of the muscle collapses and a new growth cone forms at the dorsal tip of the predominating finger. Thus, we observe that complete growth cone collapse occurs in vivo and not just in culture assays. In contrast to studies indicating that collapse occurs upon contact with repulsive substrata, collapse of the VD growth cones may result from an intrinsic signal that serves to maintain growth cone primacy and conserve cellular material.     

Phosphorylation by extracellular signal-regulated kinase of a multidomain adaptor protein, vinexin, at synapses

Vinexin is an adaptor protein that is supposed to play pivotal roles in cell adhesion, cytoskeletal organization and signaling. At least three splice variants, vinexinalpha, beta and gamma, have so far been reported. In spite of the possible importance of vinexin, the properties and functions of vinexin in neuronal cells are almost unknown. Here we show that vinexin isoforms are expressed in rat brain in a developmental stage-dependent manner, and that vinexinalpha is relatively abundant in the telencephalon regions of the adult rat brain. An immunohistochemical study showed the localization of vinexinalpha in neurons and glia in the rat brain. In primary cultured rat hippocampal neurons, vinexin was found to be present at synapses and filopodia in growth cones by immunofluorescent analyses. Biochemical fractionation revealed the distribution of vinexin in synaptosomes. Nerve terminal localization of vinexin was confirmed by electron microscopy. Vinexinbeta is reported to be phosphorylated by extracellular signal-regulated kinase (ERK) at Ser189, which is equivalent to Ser593 of vinexinalpha. We thus constructed a site- and phosphorylation state-specific antibody to monitor the ERK-mediated phosphorylation of vinexin. In immunofluorescent analyses, the phosphorylation was observed at synapses formed among cultured rat hippocampal neurons and it was reduced by treatment of the cells with PD98059. In an immunoelectron microscopic examination, the phosphorylation signal was mainly detected on the postsynaptic side of synapses in the rat hippocampal neurons. As active ERK was co-localized with vinexin in synapses, the ERK signal is likely to be involved in the regulation of vinexin-dependent cellular processes in synapses. On the other hand, the phosphorylation was hardly detected in neurons cultured for 3 days, suggesting the presence of a yet unidentified regulatory mechanism of vinexin at the growth cone.     

Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation [published erratum appears in J Cell Biol 1998 Dec 28;143(7):following 2090]

Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.     

Modulation of growth cone filopodial length by carbon monoxide

Carbon monoxide (CO) is physiologically produced via heme degradation by heme oxygenase enzymes. Whereas CO has been identified as an important physiological signaling molecule, the roles it plays in neuronal development and regeneration are poorly understood. During these events, growth cones guide axons through a rich cellular environment to locate target cells and establish synaptic connections. Previously, we have shown that another gaseous signaling molecule, nitric oxide (NO), has potent effects on growth cone motility. With NO and CO sharing similar cellular targets, we wanted to determine whether CO affected growth cone motility as well. We assessed how CO affected growth cone filopodial length and determined the signaling pathway by which this effect was mediated. Using two well‐characterized neurons from the freshwater snail, Helisoma trivolvis , it was found that the CO donor, carbon monoxide releasing molecule‐2 (CORM‐2), increased filopodial length. CO utilized a signaling pathway that involved the activation of soluble guanylyl cyclase, protein kinase G, and ryanodine receptors. While increases in filopodial length often occur from robust increases in intracellular calcium levels, the timing in which CO increased filopodial length corresponded with low basal calcium levels in growth cones. Taken together with findings of a heme oxygenase‐like protein in the Helisoma nervous system, these results provide evidence for CO as a modulator of growth cone motility and implicate CO as a neuromodulatory signal during neuronal development and/or regeneration. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 677–690, 2017