Ethanol inhibits L1-mediated neurite outgrowth in postnatal rat cerebellar granule cells

The neuropathology of the effects of ethanol on the developing central nervous system are similar to those of patients with mutations in L1, a neural cell adhesion molecule. This observation suggests that inhibition of L1 plays a role in the pathogenesis of alcohol-related neurodevelopmental disorders. Here we examine the effects of ethanol on L1 homophilic binding and on L1-mediated neurite outgrowth. Ethanol had no effect on cell adhesion or aggregation in a myeloma cell line expressing full-length human L1. In contrast, the rate of L1-mediated neurite outgrowth of rat postnatal day 6 cerebellar granule cells grown on a substratum of NgCAM, the chick homologue of L1, was inhibited by 48.6% in the presence of ethanol with a half-maximal concentration of 4.7 mM. The same effect was found with soluble L1-Fc, thus showing that the inhibitory effect is not dependent on cell adhesion. In contrast, neither laminin nor N-cadherin-mediated neurite outgrowth was inhibited by physiologic concentrations of ethanol. We conclude that one mechanism of ethanol's toxicity to the developing central nervous system may be the inhibition of L1-mediated neurite outgrowth.     

Ethanol inhibits L1 cell adhesion molecule activation of mitogen-activated protein kinases

Inhibition of the functions of L1 cell adhesion molecule (L1) by ethanol has been implicated in the pathogenesis of the neurodevelopmental aspects of the fetal alcohol syndrome (FAS). Ethanol at pharmacological concentrations has been shown to inhibit L1-mediated neurite outgrowth of rat post-natal day 6 cerebellar granule cells (CGN). Extracellular signal-related kinases (ERK) 1/2 activation occurs following L1 clustering. Reduction in phosphoERK1/2 by inhibition of mitogen-activated protein kinase kinase (MEK) reduces neurite outgrowth of cerebellar neurons. Here, we examine the effects of ethanol on L1 activation of ERK1/2, and whether this activation occurs via activation of fibroblast growth factor receptor 1 (FGFR1). Ethanol at 25 mm markedly inhibited ERK1/2 activation by both clustering L1 with cross-linked monoclonal antibodies, or by L1-Fc chimeric proteins. Clustering L1 with subsequent ERK1/2 activation did not result in tyrosine phosphorylation of the FGFR1. In addition, inhibition of FGFR1 tyrosine kinase blocked basic fibroblast growth factor (bFGF) activation of ERK1/2, but did not affect activation of ERK1/2 by clustered L1. We conclude that ethanol disrupts the signaling pathway between L1 clustering and ERK1/2 activation, and that this occurs independently of the FGFR1 pathway in cerebellar granule cells.     

Phospholipase D2 functions as a downstream signaling molecule of MAP kinase pathway in L1-stimulated neurite outgrowth of cerebellar granule neurons

Stimulation of the neuronal cell adhesion molecule L1 in cerebellar granule neurons (CGNs) enhances neurite outgrowth and this response is inhibited by the primary alcohol ethanol. Because primary alcohols suppress the formation of the signaling lipid phosphatidic acid (PA) by phospholipase D (PLD), this observation prompted us to investigate whether PLD plays a role in the L1-mediated neurite outgrowth in CGNs. In the cerebellum of postnatal day 8 mice, PLD2 protein was abundantly expressed, while PLD1 expression was not detected. The L1-stimulated neurite outgrowth was inhibited by primary alcohols and by overexpression of lipase-deficient PLD2. Increases in cellular PA levels by direct PA application or overexpression of wild-type PLD2 mimicked the L1-dependent stimulation of neurite outgrowth. Furthermore, it was found that L1 stimulation in CGNs increased PLD activity concomitantly with phosphorylation of extracellular signal-regulated kinase (ERK), both of which were inhibited by the MAP kinase-ERK kinase (MEK) inhibitor. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK to mediate the L1-dependent neurite outgrowth of CGNs, a mechanism that may be related to alcohol-related neurodevelopmental disorders.     

神经细胞粘附分子通过P21活化激酶1促进神经突生长

目的探索神经细胞粘附分子(NCAM)促进神经突生长的分子机制。方法对新生小鼠脑组织行免疫共沉淀以筛选NCAM的结合伴侣。向体外培养的海马神经元中加入免疫共沉淀的阳性筛选分子的抑制剂,观察其对NCAM促进神经突生长作用的影响。提取新生小鼠脑内生长锥以及脂筏,检测NCAM、NCAM的结合伴侣及其上、下游分子在小鼠脑内的空间分布。结果免疫共沉淀发现P21活化激酶1(Pak1)为NCAM的结合伴侣,Pak1抑制剂可以阻断NCAM促进神经突生长的作用。对小鼠脑内脂筏的研究发现NCAM和Pak1上游激活物Pak相互作用交换因子(PIX)、细胞分裂周期蛋白42(Cdc42)在生长锥脂筏上富集,提示NCAM与Pak1的结合以及Pak1的活化可能在脂筏上完成。结论 NCAM通过Pak1途径促进神经突生长,且这一作用的实现可能依赖于脂筏。     

Ethanol inhibits L1 cell adhesion molecule tyrosine phosphorylation and dephosphorylation and activation of pp60src

Fetal alcohol syndrome is a leading cause of mental retardation. The neuropathology found in patients with fetal alcohol syndrome overlaps with those with mutations in the gene for cell adhesion molecule (L1). We have previously shown that L1-mediated neurite outgrowth and L1 activation of extracellular receptor kinases 1/2 are inhibited at low concentrations of ethanol. One possible mechanism for this effect is through disruption of a tyrosine-based sorting signal, Y(1176)RSLE, on the cytoplasmic domain of L1. Our goal was to determine if ethanol inhibited the sorting signal or its phosphorylation state. Using cerebellar granule neurons and dorsal root ganglion neurons, we found that ethanol had no effect on L1 distribution to the growth cone or its ability to be expressed on the cell surface as determined by confocal microscopy. In cerebellar granule neurons, clustering of L1 resulted in increased dephosphorylation of Y(1176), increased L1 tyrosine phosphorylation, and an increase in the activation of pp60^src as measured by immunoblot. All changes were inhibited by 25 mM ethanol. Using PP2 to inhibit pp60^src activation resulted in inhibition of increases in L1 tyrosine and extracellular receptor kinases 1/2 phosphorylation, and Y(1176) dephosphorylation. We conclude that ethanol disrupts L1 trafficking/signaling following its expression on the surface of the growth cone, and prior to its activation of pp60^src.     

Neurite Outgrowth is Dependent on the Association of c-Src and Lipid Rafts

Regulation of neurite outgrowth is an important aspect not only for proper development of the nervous system but also for tissue regeneration after nerve injury and the treatment of neuropathological conditions. Here, we report that neurite outgrowth in cortical neuron and neuro 2A (N2A) cell was dependent on intact lipid rafts, as well as the enhanced localization of c-Src in the lipid rafts. Src inhibition or lipid rafts disruption could specifically block c-Src phosphorylation profile, pY416 Src increase and pY529 Src decrease, they also resulted in pY529 Src and c-terminal Src kinase (Csk) partition out of lipid rafts. Thus, we concluded that c-Src signal cascades within the lipid rafts is crucial for efficient neurite outgrowth.     

Segregation of Nogo66 receptors into lipid rafts in rat brain and inhibition of Nogo66 signaling by cholesterol depletion

NogoA, a myelin-associated component, inhibits neurite outgrowth. Nogo66, a portion of NogoA, binds to Nogo66 receptor (NgR) and induces the inhibitory signaling. LINGO-1 and p75 neurotrophin receptor (p75), the low-affinity nerve growth factor receptor, are also required for NogoA signaling. However, signaling mechanisms downstream to Nogo receptor remain poorly understood. Here, we observed that NgR and p75 were colocalized in low-density membrane raft fractions derived from forebrains and cerebella as well as from cerebellar granule cells. NgR interacted with p75 in lipid rafts. In addition, disruption of lipid rafts by beta-methylcyclodextrin, a cholesterol-binding reagent, reduced the Nogo66 signaling. Our results suggest an important role of lipid rafts in facilitating the interaction between NgRs and provide insight into mechanisms underlying the inhibition of neurite outgrowth by NogoA.     

GRIN1 Regulates ��-Opioid Receptor Activities by Tethering the Receptor and G Protein in the Lipid Raft

The lipid raft location of μ-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein α-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulldown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence ²⁶⁷GSKEK²⁷¹ within the MOR third intracellular loop that is not involved in Gα interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Gα interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Gα. Furthermore, overexpression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-β-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Gα with MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth processes.     

Lipid Raft-dependent Endocytosis of Close Homolog of Adhesion Molecule L1 (CHL1) Promotes Neuritogenesis

CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. We report here that neuritogenesis-promoting ligand-dependent cell surface clustering of CHL1 induces palmitoylation and lipid raft-dependent endocytosis of CHL1. We identify βII spectrin as a binding partner of CHL1, and we show that partial disruption of the complex between CHL1 and βII spectrin accompanies CHL1 endocytosis. Inhibition of the association of CHL1 with lipid rafts by pharmacological disruption of lipid rafts or by mutation of cysteine 1102 within the intracellular domain of CHL1 reduces endocytosis of CHL1. Endocytosis of CHL1 is also reduced by nifedipine, an inhibitor of the L-type voltage-dependent Ca²⁺ channels. CHL1-dependent neurite outgrowth is reduced by inhibitors of lipid raft assembly, inhibitors of voltage-dependent Ca²⁺ channels, and overexpression of CHL1 with mutated cysteine Cys-1102. Our results suggest that ligand-induced and lipid raft-dependent regulation of CHL1 adhesion via Ca²⁺-dependent remodeling of the CHL1-βII spectrin complex and CHL1 endocytosis are required for CHL1-dependent neurite outgrowth.     

Nerve growth factor stimulates the concentration of TrkA within lipid rafts and extracellular signal-regulated kinase activation through c-Cbl-associated protein

Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation.     

Cholesterol-mediated neurite outgrowth is differently regulated between cortical and hippocampal neurons

The acquisition of neuronal type-specific morphogenesis is a central feature of neuronal differentiation and has important consequences for region-specific nervous system functions. Here, we report that the cell type-specific cholesterol profile determines the differential modulation of axon and dendrite outgrowths in hippocampal and cerebral cortical neurons in culture. The extent of axon and dendrite outgrowths is greater and the polarity formation occurs earlier in cortical neurons than in hippocampal neurons. The cholesterol concentrations in total homogenate and the lipid rafts from hippocampal neurons are significantly higher than those from cortical neurons. Cholesterol depletion by beta-cyclodextrin markedly enhanced the neurite outgrowth and accelerated the establishment of neuronal polarity in hippocampal neurons, which were similarly observed in nontreated cortical neurons, whereas cholesterol loading had no effects. In contrast, both depletion and loading of cholesterol decreased the neurite outgrowths in cortical neurons. The stimulation of neurite outgrowth and polarity formation induced by cholesterol depletion was accompanied by an enhanced localization of Fyn, a Src kinase, in the lipid rafts of hippocampal neurons. A concomitant treatment with beta-cyclodextrin and a Src family kinase inhibitor, PP2, specifically blocked axon outgrowth but not dendrite outgrowth (both of which were enhanced by beta-cyclodextrin) in hippocampal neurons, suggesting that axon outgrowth modulated by cholesterol is induced in a Fyn-dependent manner. These results suggest that cellular cholesterol modulates axon and dendrite outgrowths and neuronal polarization under culture conditions and also that the difference in cholesterol profile between hippocampal and cortical neurons underlies the difference in neurite outgrowth between these two types of neurons.     

Effects of ethanol and NAP on cerebellar expression of the neural cell adhesion molecule L1

The neural cell adhesion molecule L1 is critical for brain development and plays a role in learning and memory in the adult. Ethanol inhibits L1-mediated cell adhesion and neurite outgrowth in cerebellar granule neurons (CGNs), and these actions might underlie the cerebellar dysmorphology of fetal alcohol spectrum disorders. The peptide NAP potently blocks ethanol inhibition of L1 adhesion and prevents ethanol teratogenesis. We used quantitative RT-PCR and Western blotting of extracts of cerebellar slices, CGNs, and astrocytes from postnatal day 7 (PD7) rats to investigate whether ethanol and NAP act in part by regulating the expression of L1. Treatment of cerebellar slices with 20 mM ethanol, 10(-12) M NAP, or both for 4 hours, 24 hours, and 10 days did not significantly affect L1 mRNA and protein levels. Similar treatment for 4 or 24 hours did not regulate L1 expression in primary cultures of CGNs and astrocytes, the predominant cerebellar cell types. Because ethanol also damages the adult cerebellum, we studied the effects of chronic ethanol exposure in adult rats. One year of binge drinking did not alter L1 gene and protein expression in extracts from whole cerebellum. Thus, ethanol does not alter L1 expression in the developing or adult cerebellum; more likely, ethanol disrupts L1 function by modifying its conformation and signaling. Likewise, NAP antagonizes the actions of ethanol without altering L1 expression.     

Association of GPI-Anchored Protein TAG-1 with Src-Family Kinase Lyn in Lipid Rafts of Cerebellar Granule Cells

We have demonstrated that antibody-mediated crosslinking of GPI-anchored TAG-1 induced activation of src-family kinase Lyn and rapid tyrosine phosphorylation of an 80-kDa protein (p80), a putative substrate for Lyn, in the lipid raft fraction prepared from primary cerebellar cultures, suggesting the functional association of TAG-1 with Lyn in lipid rafts of the rat cerebellum. In this study, the association was confirmed using a cDNA expression system. TAG-1-expressing CHO transfectants exhibited enhanced self-aggregation and promoted neurite outgrowth of primary cerebellar cultures as a culture substrate. The anti-TAG-1 antibody co-immunoprecipitated Lyn with TAG-1 and induced co-patching of TAG-1 with Lyn in both TAG-1 and Lyn-expressing CHO transfectants. Density gradient analysis revealed that TAG-1 is present in the lipid raft fraction of the CHO transfectants. Furthermore, pretreatment with a sphingolipid biosynthesis inhibitor ISP-1 reduced the extent of tyrosine phosphorylation of p80 by the antibody-mediated crosslinking of TAG-1. Immunocytochemical study showed that both TAG-1 and Lyn are present in cerebellar granule cells. These observations suggest that TAG-1 associates with Lyn in lipid rafts of rat cerebellar granule cells.     

Binding of soluble myelin-associated glycoprotein to specific gangliosides induces the association of p75NTR to lipid rafts and signal transduction

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. Here we show that gangliosides, GT1b and GD1a, as well as the Nogo receptor, are functional binding partners for soluble MAG-Fc. Postnatal cerebellar neurons from mice deficient in the GalNAcT gene are insensitive to MAG with regard to neurite outgrowth and lack in the activation of RhoA. MAG-Fc or the antibody to GT1b and GD1a elicits recruitment of p75(NTR.) to lipid rafts, specialized microdomain for signal transduction. Disruption of lipid rafts results in abolishment of inhibitory effects of MAG-Fc and the Nogo peptide. These findings establish gangliosides as functional binding partners for soluble MAG. Gangliosides may play a role in translocation of p75(NTR.) to lipid rafts for initiation of the signal transduction.     

Distinct functions of Trio GEF domains in axon outgrowth of cerebellar granule neurons

As a critical guanine nucleotide exchange factor (GEF) regulating neurite outgrowth, Trio coordinates multiple processes of cytoskeletal dynamics through activating Rac1, Cdc42 and RhoA small GTPases by two GEF domains, but the in vivo roles of these GEF domains and corresponding downstream effectors have not been determined yet. We established multiple lines of knockout mice and assessed the respective roles of Trio GEF domains and Rac1 in axon outgrowth. Knockout of total Trio in cerebellar granule neurons (CGNs) led to an impaired F-actin rearrangement of growth cone and hence a retarded neurite outgrowth. Such a retardation was reproduced by inhibition of GEF1 domain or knockdown of Cdc42 and restored apparently by introduction of active Cdc42. As Rac1 deficiency did not affect the neurite outgrowth of CGNs, we suggested that Trio GEF1-mediated Cdc42 activation was required for neurite outgrowth. We established a GEF2-knockout line with deletion of all Trio isoforms except a cerebella-specific Trio8, a short isoform of Trio without GEF2 domain, and used this line as a GEF2-deficient animal model. The GEF2-deficient CGNs had a normal neurite outgrowth but abolished Netrin-1-promoted growth, without affecting Netrin-1 induced Rac1 activation. We thus suggested that Trio GEF1-mediated Cdc42 activation rather than Rac1 activation drives the F-actin dynamics necessary for neurite outgrowth, while GEF2 functions in Netrin-1-promoted neurite elongation. Our results delineated the distinct roles of Trio GEF domains in neurite outgrowth, which is instructive to understand the pathogenesis of clinical Trio-related neurodevelopmental disorders.     

Regulation of growth cone extension by SNARE proteins.

Recent studies have suggested that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion with the surface plasma membrane, which leads to neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. We studied this mechanism by overexpressing GFP-fusion SNAREs including VAMP-2, SNAP-25A, and syntaxin1A in PC12 cells to investigate the role of SNAREs in neurite outgrowth. When overexpressed in PC12 cells, VAMP-2 promoted neurite elongation, whereas SNAP-25A stimulated neurite sprouting. On the other hand, overexpression of syntaxin1A neither promoted nor inhibited neurite outgrowth. Thus, VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.     

Extracellular matrix molecules and cell adhesion molecules induce neurites through different mechanisms

It has recently become clear that both extracellular matrix (ECM) glycoproteins and various cell adhesion molecules (CAMs) can promote neurite outgrowth from primary neurons, though little is known of the intracellular mechanisms through which these signals are transduced. We have previously obtained evidence that protein kinase C function is an important part of the neuronal response to laminin (Bixby, J.L. 1989. Neuron. 3:287-297). Because such CAMs as L1 (Lagenauer, C., and V. Lemmon. 1987. Proc. Natl. Acad. Sci. USA. 84:7753-7757) and N-cadherin (Bixby, J.L. and R. Zhang. 1990. J. Cell Biol. 110:1253-1260) can be purified and used as substrates to promote neurite growth, we have now tested whether the response to CAMs is similarly dependent on protein kinase C. We find that inhibition of protein kinase C inhibits growth on fibronectin or collagen as well as on laminin. In contrast, C kinase inhibition actually potentiates the initial growth response to L1 or N- cadherin. The later "phase" of outgrowth on both of these CAMs is inhibited, however. Additionally, phorbol esters, which have no effect on neurite growth when optimal laminin concentrations are used, potentiate growth even on optimal concentrations of L1 or N-cadherin. The results indicate that different intracellular mechanisms operate during initial process outgrowth on ECM substrates as compared to CAM substrates, and suggest that protein kinase C function is required for continued neurite growth on each of these glycoproteins.     

L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth

The immunoglobulin superfamily recognition molecule L1 plays important functional roles in the developing and adult nervous system. Metalloprotease-mediated cleavage of this adhesion molecule has been shown to stimulate cellular migration and neurite outgrowth. We demonstrate here that L1 cleavage is mediated by two distinct members of the disintegrin and metalloprotease family, ADAM10 and ADAM17. This cleavage is differently regulated and leads to the generation of a membrane bound C-terminal fragment, which is further processed through gamma-secretase activity. Pharmacological approaches with two hydroxamate-based inhibitors with different preferences in blocking ADAM10 and ADAM17, as well as loss of function and gain of function studies in murine embryonic fibroblasts, showed that constitutive shedding of L1 is mediated by ADAM10 while phorbol ester stimulation or cholesterol depletion led to ADAM17-mediated L1 cleavage. In contrast, N-methyl-d-aspartate treatment of primary neurons stimulated ADAM10-mediated L1 shedding. Both proteases were able to affect L1-mediated adhesion and haptotactic migration of neuronal cells. In particular, both proteases were involved in L1-dependent neurite outgrowth of cerebellar neurons. Thus, our data identify ADAM10 and ADAM17 as differentially regulated L1 membrane sheddases, both critically affecting the physiological functions of this adhesion protein.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts

Although both c-Ret and GFRalpha1 are required for responsiveness to GDNF, GFRalpha1 is widely expressed in the absence of c-Ret, suggesting alternative roles for "ectopic" sites of GFRalpha1 expression. We show that GFRalpha1 is released by neuronal cells, Schwann cells, and injured sciatic nerve. c-Ret stimulation in trans by soluble or immobilized GFRalpha1 potentiates downstream signaling, neurite outgrowth, and neuronal survival, and elicits dramatic localized expansions of axons and growth cones. Soluble GFRalpha1 mediates robust recruitment of c-Ret to lipid rafts via a novel mechanism requiring the c-Ret tyrosine kinase. Activated c-Ret associates with different adaptor proteins inside and outside lipid rafts. These results provide an explanation for the tissue distribution of GFRalpha1, supporting the physiological importance of c-Ret activation in trans as a novel mechanism to potentiate and diversify the biological responses to GDNF.