Research ArticleNeuroscience

Neuronal Growth Cone Retraction Relies on Proneurotrophin Receptor Signaling Through Rac

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Science Signaling  06 Dec 2011:
Vol. 4, Issue 202, pp. ra82
DOI: 10.1126/scisignal.2002060

Abstract

Growth of axons and dendrites is a dynamic process that involves guidance molecules, adhesion proteins, and neurotrophic factors. Although neurite extension is stimulated by the neurotrophin nerve growth factor (NGF), we found that the precursor of NGF, proNGF, induced acute collapse of growth cones of cultured hippocampal neurons. This retraction was initiated by an interaction between the p75 neurotrophin receptor (p75NTR) and the sortilin family member SorCS2 (sortilin-related VPS10 domain–containing receptor 2). Binding of proNGF to the p75NTR-SorCS2 complex induced growth cone retraction by initiating the dissociation of the guanine nucleotide exchange factor Trio from the p75NTR-SorCS2 complex, resulting in decreased Rac activity and, consequently, growth cone collapse. The actin-bundling protein fascin was also inactivated, contributing to the destabilization and collapse of actin filaments. These results identify a bifunctional signaling mechanism by which proNGF regulates actin dynamics to acutely modulate neuronal morphology.

Introduction

The formation of neuronal networks in the developing nervous system depends on the navigation of axons and dendrites, which is controlled by attractive and repulsive guidance cues (1, 2). Although much attention has focused on the signaling mechanisms that promote the branching and extension of dendrites and axons, the extrinsic signals that restrict the size and extent of growth of neuronal processes remain incompletely defined. In particular, the molecular mechanisms by which extrinsic signals are translated to intracellular pathways mediating retraction are not well established.

Growth cones at the tip of extending neurites are rich in actin filament–containing structures such as lamellipodia and filopodia, which in turn are stabilized by the actin-bundling protein fascin (3). The dynamic extension and retraction of these actin structures is thought to control growth cone motility and is regulated by Rho family guanosine triphosphatases (GTPases) (4).

In addition to ephrins, neuropilins, and semaphorins (5, 6), the outgrowth of dendrites and axons is influenced by neurotrophins (7, 8). Neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and NT-4, are required for neuronal survival and differentiative, synapse formation, and synaptic plasticity (9). These secreted proteins act through two classes of receptor: the tropomyosin-related kinase (Trk) receptor, which transduces survival and differentiation signals, and the p75 neurotrophin receptor (p75NTR), which encodes an intracellular death domain. Neurotrophins are synthesized as precursors, or proneurotrophins, that can be cleaved intracellularly by furin or prohormone convertases, or extracellularly by plasmin or matrix metalloproteases, to produce mature forms (1012). Mature NGF activates TrkA to mediate survival and differentiative signaling. However, proneurotrophins, including proNGF, are not inactive; they bind to a receptor complex of p75NTR and sortilin, a Vps10p (vacuolar protein sorting 10 protein) domain–containing transmembrane protein, to initiate apoptosis (10, 12, 13).

Here, we have identified proNGF as a ligand that initiates acute collapse of growth cones in hippocampal neurons. This action required p75NTR and SorCS2 (sortilin-related VPS10 domain–containing receptor 2), another Vps10p domain family member that served as a co-receptor with p75NTR. ProNGF induced displacement of the guanine nucleotide exchange factor (GEF) Trio from p75NTR and SorCS2, thereby decreasing local activity of the small GTPase Rac and filopodia formation. ProNGF binding also led to phosphorylation of the actin-bundling protein fascin by protein kinase C (PKC) and thereby to fascin inactivation and destabilization of existing actin filaments. These results thus identify two mechanisms by which an extracellular cue, proNGF, initiates intracellular pathways to regulate neuronal morphology by inducing rapid growth cone collapse.

Results

ProNGF induces growth cone collapse

Neurotrophins have been classically defined as trophic factors involved in growth cone turning and extension (14). However, treatment of primary mouse hippocampal neurons with 0.1 nM proNGF led to rapid freezing of growth cone movement, followed by growth cone collapse over a period of minutes. We imaged actin dynamics in live neurons using a peptide that labels filamentous actin (F-actin) without disrupting the cytoskeleton (15). Under basal conditions, filopodial movement in the growth cone was dynamic (Fig. 1A, movie S1, and fig. S1). However, in a subset (~25%) of neurons, the motion of the growth cone froze within 2 min of proNGF addition, and the now rigid actin structures subsequently collapsed. This collapse was not limited to LifeAct-labeled filaments, as judged by differential interference contrast (DIC) microscopy images or by staining for endogenous actin, fascin, and p75NTR (Fig. 1B, lower panel, and fig. S1). The remaining neurons (~75%) were unaffected by proNGF.

Fig. 1

ProNGF signaling through p75NTR and SorCS2 induces growth cone retraction. (A) E15.5 DIV3 hippocampal neurons transfected with LifeAct-RFP (red fluorescent protein) were imaged by time-lapse microscopy before (left panels) or after (right panels) treatment with proNGF. Time is indicated in (min:s). See also movie S1. (B) Neurons were incubated with proNGF for 20 min, fixed, and stained for actin, fascin, and p75NTR. Arrows indicate collapsed growth cones; asterisks indicate intact growth cones. Insets 1 and 2 are provided at higher magnification. (C) Quantification of (B). Control, untreated controls; proNGF, cells exposed to proNGF (10 ng/ml) (n = 12 independent experiments); NGF, cells incubated with NGF (5 ng/ml) (n = 4 independent experiments). ***P < 0.001; n.s., not significant; one-way analysis of variance (ANOVA). (D) Neurons were fixed and stained for p75NTR and SorCS2. Arrows highlight growth cones. (E) Cos-7 cells were transfected with indicated constructs, and lysates were immunoprecipitated with anti-HA antibodies and analyzed by Western blot (WB) with indicated antibodies. n = 4 independent experiments. (F) Neurons were preincubated with anti-SorCS2 antibodies or control IgG before addition of proNGF for 20 min, and collapse was quantified. n = 4 independent experiments. ***P < 0.001; n.s., not significant; one-way ANOVA. Scale bars, 10 μm [(A) and (B)] and 20 μm (D).

ProNGF binds to a p75NTR and sortilin receptor complex (10, 12). To determine whether the subpopulation of cells responsive to proNGF had these receptors, we treated cultures with proNGF for 20 min and then fixed and stained the cells for endogenous actin, the actin-bundling protein fascin, and p75NTR. Untreated cells displayed extended, fan-like growth cones and filopodia rich in actin and fascin (Fig. 1, B, top panel, and C). Cells positive for p75NTR responded to proNGF treatment with growth cone collapse (Fig. 1, B, lower panel, arrows, and C). Growth cones of p75NTR-negative cells did not collapse after addition of proNGF, however, nor did NGF elicit actin rearrangement in p75NTR-positive cells (Fig. 1, B, lower panel, asterisks, and C). Cultures from p75-deficient embryos failed to respond to proNGF (fig. S2A), indicating that p75NTR was required for proNGF-mediated collapse of actin- and fascin-rich protrusions.

We were not able to detect sortilin in neurons of this age [neurons collected at embryonic day 15 (E15) and stained at 3 days in vitro (DIV3); fig. S2, B and C]. However, we detected a related protein, SorCS2, in p75NTR-positive cells in vitro (Fig. 1D and fig. S2C) and in the hippocampus (fig. S2D), and immunoprecipitation of transfected cells indicated that, like sortilin, SorCS2 interacted with proNGF (Fig. 1E). To determine whether SorCS2 was involved in retraction of actin-rich structures, we preincubated cultures with an antibody directed against the SorCS2 ectodomain before adding proNGF. Then, we fixed the cells and visualized actin, fascin, and p75NTR localization. Preincubation with anti-SorCS2 had no independent effect on neuronal morphology, but it blocked the ability of proNGF to induce growth cone collapse, whereas control immunoglobulin G (IgG) or antibodies directed against the sortilin ectodomain did not impair the proNGF response (Fig. 1F and fig. S2E). These data suggest that p75NTR and SorCS2 act as co-receptors for proNGF to mediate acute alterations in actin morphology.

Trio interacts with the p75NTR and SorCS2 receptor complex

To identify signaling proteins downstream of the proNGF-activated p75NTR and SorCS2 complex, we performed a proteomic analysis of proteins that specifically associated with p75NTR and the closely related SorCS2 family member, sortilin, as compared to p75NTR alone. Among the proteins detected by mass spectrometry, we identified Trio, a scaffold protein containing Rac and Rho GEF domains (16) (Fig. 2A and fig. S3). We confirmed the interaction between p75NTR and Trio by coimmunoprecipitation. Coexpression of p75NTR and sortilin or p75NTR and SorCS2 in cells containing Trio or individual Trio domains resulted in a stable complex containing p75NTR and Trio (Fig. 2B), an interaction that required the Trio putative serine and threonine kinase (kin) domain (Fig. 2, C to E). To determine whether ligand binding altered the interaction of Trio with p75NTR and SorCS2, we treated cells coexpressing these receptors with proNGF and then immunoprecipitated the lysates with anti-p75NTR. Addition of proNGF markedly reduced the basal interaction of p75NTR with Trio (Fig. 2, F and G), indicating that proNGF induces the displacement of Trio from the p75NTR and SorCS2 complex.

Fig. 2

Trio interacts with the p75NTR and SorCS2 receptor complex. (A) Coomassie Blue staining of proteins coprecipitating with p75NTR in HT1080 cells transfected with constructs encoding p75NTR and sortilin. Trio was identified by subsequent mass spectrometry (see also fig. S3). (B) Coimmunoprecipitation of endogenous Trio with p75NTR from HT1080 cells expressing both p75NTR and sortilin or p75NTR and SorCS2, but not p75NTR alone. Input, cell lysate before immunoprecipitation (IP). n = 5 independent experiments. (C and D) Myc-tagged Trio domains were coexpressed with HA-p75NTR in 293T cells, immunoprecipitated with anti-Myc (C) or with anti-HA (D) antibodies, and Western blots were probed with the indicated antibodies. n = 5 (C) and 4 (D) independent experiments. (E) Schematic representation of full-length Trio. (F and G) HT1080 cells expressing p75NTR and SorCS2 were incubated with proNGF for 20 min and lysed, and p75NTR was immunoprecipitated. (F) Western blots were probed with indicated antibodies. (G) Coprecipitation of Trio with p75NTR was quantified from 10 separate experiments by densitometry and normalized to the input. ***P < 0.001, Student’s t test.

In hippocampal neurons, endogenous Trio localized to actin-rich protrusions (Fig. 3A, top panel, and fig. S4A), as previously shown (17). ProNGF led to the loss of Trio from the neurite tip in p75NTR-positive cells (Fig. 3A, bottom panel, arrows), whereas Trio remained enriched in intact growth cones of p75NTR-negative cells (Fig. 3A, bottom panel, asterisk). We confirmed the presence of the p75NTR, SorCS2, and Trio trimeric complex in embryonic rat brain lysates in vivo (Fig. 3B). We expressed the Trio kinase domain, or a kinase-dead mutant (trio kinK2921A; fig. S4B), in cultured hippocampal neurons to displace endogenous Trio (and thereby its GEF1 and GEF2 activities) from the p75NTR complex. This led to collapse of actin structures in the absence of proNGF (Fig. 3, C and D), indicating that displacement of Trio is sufficient to induce growth cone collapse.

Fig. 3

Dissociation of Trio from the p75NTR and SorCS2 complex leads to growth cone collapse. (A) Hippocampal neurons were incubated with proNGF for 20 min, fixed, and stained for Trio, actin, and p75NTR. (B) Endogenous Trio and SorCS2 form a complex with p75NTR. Embryonic brain lysates were immunoprecipitated with antibodies against p75NTR or control IgGs and probed for indicated proteins. n = 4 independent experiments. (C and D) Hippocampal neurons were transfected at DIV2 with the Trio kinase domain containing an IRES-GFP element or the kinase-dead mutant K2921A and fixed at DIV3. (C) Growth cone collapse was quantified, scoring 47 (Trio-kin) and 50 (kinK2921A) neurites in three independent experiments. ***P < 0.001; n.s., not significant; one-way ANOVA. (D) Arrows indicate collapsed growth cones. Scale bars, 10 μm.

ProNGF binding leads to Rac inactivation

The Drosophila Trio protein promotes axon growth and guidance through its ability to act as a GEF for Rac (18). To assess whether proNGF-induced growth cone collapse is a consequence of the inactivation of Trio and thereby Rac, we used the Cdc42/Rac interactive binding (CRIB) domain of the Rac effector p21-activated kinase (PAK-CRIB) to isolate and purify activated Rac (19). ProNGF exposure led to a significant decrease in Rac activity in primary hippocampal neurons compared to that in untreated neurons (Fig. 4, A and B). To determine whether decreased Rac activity resulted in growth cone collapse, we incubated hippocampal neurons with the Rac inhibitor EHT 1864 or the Trio GEF1 domain inhibitor ITX3 (20). Both drugs markedly decreased Rac activity in primary neuronal cultures (fig. S5A) and promoted growth cone collapse in all treated cells (Fig. 4C), consistent with the notion that Rac inactivation may underlie the proNGF-induced retraction of actin-rich structures. Finally, we evaluated whether expression of the Trio GEF1 domain, which promotes Rac activation, could overcome proNGF-induced growth cone collapse. Indeed, expression of the Trio GEF1 domain abolished proNGF-induced growth cone retraction (Fig. 4, D and E). In contrast, inactivation of RhoA with C3 transferase or exposure to the ROCK (Rho-associated kinase) inhibitor Y27632 neither induced growth cone collapse nor did these treatments interfere with proNGF-mediated growth cone collapse (Fig. 4F and fig. S5, B and C). Together, these results suggest that proNGF binding to the p75NTR and SorCS2 receptor complex leads to displacement of Trio, reduced activation of Rac, and collapse of actin-rich protrusions.

Fig. 4

Decreased Rac activity underlies growth cone collapse. (A) Hippocampal neurons were incubated with proNGF for 20 min, and cell lysates were incubated with GST–PAK-CRIB beads to isolate activated Rac. (B) Isolated activated Rac was measured by densitometry and normalized to total Rac in the input. Shown is the mean ± SEM of four independent experiments. *P = 0.018; Student’s t test. (C) Inhibition of Rac or Trio GEF1 activities induces growth cone collapse. Hippocampal neurons were treated with EHT 1864 or ITX3, fixed, and stained for actin. Arrows indicate collapsed growth cones. n = 3 independent experiments. (D) Expression of the Trio GEF1 domain rescues proNGF-induced collapse. Hippocampal neurons were transfected with the Trio GEF1 domain containing an IRES-GFP element at DIV2. The following day, cells were treated with proNGF, fixed, and stained with indicated antibodies. (E) Quantitation of (D). Shown is the mean ± SEM of four independent experiments; ***P < 0.001; n.s., not significant; Student’s t test. (F) Inhibition of RhoA activity does not prevent proNGF-induced growth cone collapse. Neurons were pretreated with C3 transferase for 4 hours or Y27632 for 45 min before proNGF exposure for 20 min, fixed, and stained; collapse was quantified in three independent experiments. Scale bars, 10 μm.

ProNGF-dependent collapse requires fascin phosphorylation

Live imaging of neurons treated with proNGF revealed an initial reduction in the extension and retraction of actin filaments, which was followed by their overt collapse (Fig. 1A, fig. S1, and movie S1). Therefore, we investigated a potential role for the actin filament stabilizing protein, fascin, in mediating growth cone retraction. Fascin is highly enriched in growth cones (3), although the mechanisms that regulate fascin bundling of filamentous actin in neurons are unclear. However, fascin interacts directly with p75NTR in non-neuronal cells (21), and inactivation of fascin by PKC-dependent phosphorylation at Ser39 (22) abrogates proNGF-dependent melanoma cell migration in a PKC-dependent manner (21).

We found that fascin also interacted with p75NTR in embryonic brain lysates (fig. S6). Therefore, we hypothesized that proNGF-induced fascin phosphorylation and its dissociation from actin filaments may contribute to growth cone destabilization and facilitate their collapse. To test this hypothesis, we preincubated hippocampal neurons with the PKC inhibitor Gö6976 or the small peptide PKC inhibitor 20–28 before exposing them to proNGF. In both cases, proNGF failed to induce collapse of actin-rich structures (Fig. 5, A and C), suggesting that PKC-dependent phosphorylation was required for this process. To confirm whether fascin was the PKC target, we expressed fascin, or a fascin phosphorylation mutant [fascinS(36,38,39)A, in which serines 36, 38, and 39 are substituted with alanines] that mimics the active, actin-bundling form of the protein (23). Addition of proNGF to hippocampal neurons expressing fascinS(36,38,39)A failed to induce growth cone collapse, whereas overexpression of wild-type fascin did not interfere with the proNGF response (Fig. 5, B and C). This suggests that fascin inactivation contributes to growth cone collapse downstream of proNGF.

Fig. 5

PKC-dependent fascin inactivation contributes to growth cone retraction. (A) Hippocampal neurons were pretreated with the PKC inhibitor Gö6976, followed by addition of proNGF, fixation, and staining. (B) Neurons were cotransfected with GFP and fascinS(36,38,39)A and 24 hours later were treated with proNGF, fixed, and stained for fascin, actin, and p75NTR. (C) Quantification of proNGF-induced collapse in cells pretreated with Gö6976, the inhibitory peptide 20–28, or expressing fascin or fascinS(36,38,39)A. Shown is the mean ± SEM of at least three independent experiments. ***P < 0.001; n.s., not significant; one-way ANOVA. Scale bars, 10 μm. (D) Model of acute proNGF action on actin dynamics. The p75NTR and SorCS2 receptor complex is associated with the Rac GEF Trio and localizes Rac activity (dark orange ovals: active Rac; light orange ovals: inactive Rac) to structures in dynamically expanding growth cones. Upon proNGF binding, Trio dissociates from the p75NTR-SorCS2 complex and Rac activity decreases to impair filopodial formation. In parallel, PKC is activated and phosphorylates and inactivates the actin-bundling protein fascin (blue circles). This leads to a destabilization of existing actin filaments and their collapse.

Collectively, these observations suggest that proNGF signaling through the p75NTR and SorCS2 receptor complex elicits rapid growth cone collapse. Growth cone collapse is mediated by the dissociation of Trio from a p75NTR and SorCS2 complex to diminish Rac activity and by the phosphorylation of fascin, leading to its dissociation from actin filaments in the growth cone (Fig. 5D). We therefore propose that proNGF uses dual, synchronized mechanisms to elicit neurite retraction by stimulating growth cone collapse.

Discussion

Here, we showed that the precursor protein form of NGF acutely affects neuronal morphology. Remodeling of neuronal processes by proNGF-induced activation of p75NTR differs from the effects of mature NGF, which promote neurite elongation through TrkA. Although many growth factors are produced as precursor proteins, the differential biological functions of the precursor and mature forms of growth factors are still poorly defined. Our results imply that conversion of proneurotrophins to mature forms may control acute morphological responses in central neurons.

The proNGF receptor p75NTR acts as a co-receptor for multiple partners, including Trk receptors, the Nogo receptor, ephrin A, and sortilin (24). Of these, sortilin is primarily an intracellular sorting receptor, which only localizes efficiently to the cell membrane in the presence of NRH2 (25). Therefore, the presence and localization of different co-receptors with p75NTR, permitting the binding of different ligands, can lead to diverse biological outcomes such as apoptosis, survival (10, 13, 24), and process retraction that depend on the selective activation of distinct signaling mechanisms. Here, we identified two signaling mechanisms downstream of a complex of the p75NTR and the sortilin family member SorCS2 that act to reduce process outgrowth. ProNGF induced dissociation of Trio from p75NTR and SorCS2 to decrease Rac activity. Concomitantly, PKC is activated in an unknown manner to induce fascin phosphorylation and its dissociation from actin filaments, permitting rapid collapse of the growth cone (Fig. 5D). Trio can promote axon guidance and enhance neurite outgrowth by coupling to netrin and DCC (deleted in colorectal cancer) (26), or by associating with Trk receptors through the scaffolding protein ankyrin-rich membrane spanning protein (ARMS) (Kidins220) to promote Rac activation (27). Here, we observe that existing complexes of Trio with p75NTR and SorCS2 undergo proNGF-induced dissociation to decrease Rac activation at the leading edges of growth cones, resulting in growth cone retraction. Together, these data indicate that Trio localization provides a switch to facilitate, or impede, filopodial extension at the growth cone in response to mature or proneurotrophins, respectively. Trio is a modular protein containing spectrin repeats that mediate protein-protein interactions, such as with ARMS (Kidins220), a GEF1 domain that can stimulate RhoG and Rac to facilitate axon outgrowth, a GEF2 domain that can stimulate RhoA to limit neurite outgrowth, and a kinase domain with poorly defined substrates (Fig. 2E) (16, 28). Hence, Trio may act as a platform to locally modulate Rac and RhoA activity in response to neurotrophins, wherein the mature form of NGF preferentially activates TrkA to promote the interaction of ARMS and Trio at the plasma membrane and Rac-dependent neurite outgrowth (27). Conversely, proNGF induces the dissociation of Trio from p75NTR to inactivate Rac locally and initiates growth cone retraction. Thus, recruitment of Trio to the plasma membrane by Trk promotes actin filament extension, whereas dissociation of Trio from p75NTR precipitates growth cone collapse. This provides an attractive model to regulate Rac activation and inactivation locally through differential use of neurotrophin ligands.

The finding that p75NTR activation by proNGF also promoted fascin phosphorylation and its dissociation from actin filaments provides a dual mechanism to induce growth cone collapse. Dephosphorylated fascin bundles actin into stable filaments to permit filopodial extension (23), and fascin decorates the entire length of filamentous actin in the growth cone (3). Fascin cycles rapidly between the actin-bound, dephosphorylated state and the actin-dissociated phosphorylated state (23). Previous studies have shown that p75NTR binds fascin (21), that PKC mediates fascin phosphorylation (22), and that PKC activation induces collapse of filopodia (29). Our current study provides a mechanistic link by which p75NTR alters fascin localization and phosphorylation to permit rapid growth cone collapse.

Our data indicate that the precise repertoire of p75NTR co-receptors on a growth cone, combined with exposure to particular ligands, will determine whether a collapse response occurs. Previous studies suggest that p75NTR acts as a signaling partner with the semaphorin receptor plexin A3 or the ephrin receptor EphB to promote growth cone collapse in sympathetic axons, through activation of the Rho-ROCK pathway (30). However, in sympathetic axons, the binding of mature BDNF to p75NTR alone is insufficient to promote acute collapse and requires the concomitant activity of the ligands Sema3 or ephrin B2 (30). In contrast, our results indicate that engagement of p75NTR and SorCS2 by proNGF is sufficient to mediate acute collapse, by means of the inactivation of Rac and fascin. The inability of mature NGF to induce collapse of hippocampal processes suggests that simultaneous engagement of p75NTR and SorCS2 by proNGF, as compared to mature NGF, results in their differential signaling to the actin cytoskeleton so that only proNGF induces retraction.

The abundance of proNGF and p75NTR is frequently increased under pathological conditions, such as under conditions of acute axonal injury, seizures, or spinal cord injury, which result in acute degeneration of neuronal projections. Our data provide insights into the mechanisms whereby neurite regrowth is impaired after acute injury, or in neurodegenerative disease states, such as Alzheimer’s disease (3133). The widespread increase in the abundance of p75NTR in injured neurons, together with increase in the abundance of proNGF that occurs in acute and chronic central nervous system injury, suggests that p75NTR-mediated axonal and dendritic degeneration may play a role in disease pathogenesis.

Materials and Methods

The LifeAct sequence (15) was cloned into the pEGFP-N1 backbone (Clontech) using the Bgl II and Bam HI restriction sites. Trio constructs containing an internal ribosomal entry site–green fluorescent protein (IRES-GFP) element were described previously (34); human sortilin complementary DNA (cDNA) or human SorCS2 cDNA was subcloned into the pcDNA3.1 hygro expression vector (Invitrogen). A Myc tag was inserted three residues after the furin site by polymerase chain reaction (PCR). Wild-type and fascinS(36,38,39)A cDNA was provided by X.-Y. Huang (35) and was subcloned into pcDNA 3.1 with the Bam HI and Eco RI sites.

Primary antibodies included anti-actin and anti-hemagglutinin (HA) (Sigma), anti-fascin and anti-Rac1 (Millipore), anti-p75NTR [9651 (36); R&D Systems], anti-sortilin and anti-SorCS2 (specific for the ectodomain of these receptors; R&D Systems), anti-Trio [Santa Cruz C20 and CT-35 (37), anti-Myc, and control IgGs (Santa Cruz)]. Fluorescent secondary antibodies were from Invitrogen; horseradish peroxidase–coupled secondary antibodies were from Sigma and Amersham. Anti-HA agarose was from Roche Diagnostics; anti-Myc (9E10) agarose and control IgG agarose were from Santa Cruz. All chemicals were from Sigma unless indicated otherwise. ProNGF was produced in Sf9 cells and purified as described (38).

Cell culture and transfection

Primary hippocampal neurons were isolated from E15 C57BL/6 mouse or E16 Sprague-Dawley rat embryos. Neurons were dissociated by incubation with 0.05% trypsin at 37°C for 8 min followed by trituration with fire-polished glass Pasteur pipettes. Cells plated on poly-d-lysine–coated dishes were grown in Neurobasal medium containing B27, 0.5 mM glutamine (Invitrogen), and 10 μM 5-fluorodeoxyuridine. Hippocampal neurons were transfected at DIV2 with Lipofectamine 2000 (Invitrogen). For coverslips, 0.5 μg of plasmid was mixed with 0.5 μl of Lipofectamine 2000, and for glass bottom dishes, 1.5 μg of plasmid was mixed with 1.5 μl of Lipofectamine 2000. Complexes were added for 30 to 45 min, and then neurons were placed into preconditioned medium until analysis the following day.

HT1080 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen) and transfected with Amaxa Nucleofector (Lonza) according to the manufacturer’s instructions. 293FT and Cos-7 cells were grown in DMEM supplemented with 10% fetal bovine serum and transfected with Lipofectamine 2000.

Cell treatments

To assay which pathways are involved in filopodial collapse, we added proNGF (5 to 10 ng/ml) or NGF (5 to 100 ng/ml) (Harlan) to primary hippocampal neurons for 20 min before fixation. As indicated, cells were pretreated with 100 nM Gö6976 (Calbiochem) for 1 hour (39), 5 μM EHT 1864 (40) for 30 min, or 100 nM ITX3 (20) (ChemBridge Corporation) for 45 min, or 8 μM 20–28 (Calbiochem) (41). To block SorCS2, we incubated cells with anti-SorCS2 (20 μg/ml) or control IgG for 20 min on ice before addition of proNGF at 37°C. To block RhoA activity, we preincubated neurons with cell-permeable C3 transferase (1 μg/ml; Cytoskeleton Inc.) for 4 hours or 10 μM Y27632 (Calbiochem) for 45 min before proNGF exposure.

Immunofluorescence

DIV3 hippocampal neurons were fixed in 4% paraformaldehyde and 20% sucrose or ice-cold methanol for 10 min and processed for fluorescence microscopy. In the case of paraformaldehyde fixation, the fixative was quenched with 50 mM NH4Cl in phosphate-buffered saline (PBS) for 5 min, and cells were permeabilized with 0.1% Triton X-100 in PBS for 2 min before blocking. Coverslips were blocked with 10% normal donkey serum, 2% bovine serum albumin, and 0.25% fish skin gelatin in tris-buffered saline for 30 min; incubated with primary antibodies diluted in blocking solution for 30 min; washed three times with tris-buffered saline and 0.25% fish skin gelatin; incubated with secondary antibodies mixed with Hoechst in blocking buffer for 30 min; and washed and mounted with Mowiol488. Cells were imaged with an LSM 510 laser-scanning confocal microscope equipped with a 40× Plan Neofluor [numerical aperture (NA) 1.3] DIC oil-immersion objective (Carl Zeiss Microimaging). Images were processed with LSM 510 software (Zeiss) and ImageJ (National Institutes of Health).

Live imaging and data analysis

Cells plated on gridded glass bottom dishes (MatTek Corporation) were imaged at DIV3 in phenol red–free Neurobasal medium (Invitrogen) supplemented with 30 mM Hepes-NaOH (pH 7.4) with an Olympus IX71 inverted microscope driven by IPLab software (BD Biosciences) and equipped with a 60× Plan Apo N objective (NA 1.42), a Hamamatsu EM charge-coupled device camera, and a heated stage maintained at 37°C, or driven by softWoRx software (version 3.7.1, DeltaVision) equipped with a CoolSNAP HQ2 camera (Photometrics) and an environmental chamber maintained at 37°C. Images were taken every 15 s. After live imaging, cells were fixed, counterstained for p75NTR, located by grid number, and examined for p75NTR expression. All movies were exported and processed with IPLab, softWoRx, and ImageJ software.

To quantify growth cone collapse, we scored all neurites in at least three to four fields of view per experiment and at least three independent experiments.

Coimmunoprecipitation

HT1080 cells were transfected by means of Amaxa with HA-p75NTR and myc-SorCS2. Twenty-four to 48 hours later, cells were treated with proNGF (25 ng/ml) for 20 min as indicated. Cells were lysed with lysis buffer [50 mM tris-HCl (pH 8.0), 140 mM NaCl, 2 mM EDTA, 1% NP-40, and 10% glycerol] supplemented with protease inhibitors, and complexes were immunoprecipitated with anti-HA–agarose (Roche). Beads were washed four times with lysis buffer supplemented with 500 mM NaCl.

To map the Trio domain required for the interaction with p75NTR, we transfected 293T cells with HA-p75NTR and constructs of the individual Trio domains. After 24 hours, cells were lysed, and complexes were immunoprecipitated with anti-HA agarose or anti-Myc agarose as indicated.

To immunoprecipitate endogenous complexes, we lysed embryonic brains in lysis buffer [50 mM tris-HCl (pH 8.0), 140 mM NaCl, 1% NP-40, and 10% glycerol] with a Dounce homogenizer. Lysates were cleared by centrifugation for 10 min at 20,000g and precleared on protein A beads for 45 min. Anti-p75NTR antibody (10 μg) (9651) or rabbit IgG was cross-linked to protein A beads with BS3 (Pierce), excess cross-linker was quenched with tris-HCl (pH 8.0), and complexes were immunoprecipitated from 10 mg of total protein per condition overnight and washed in lysis buffer supplemented with 400 mM NaCl. For detection of the p75NTR and fascin interaction, the membrane was cross-linked with 2.5% glutaraldehyde in PBS for 30 min after transfer and before blocking to minimize background from the antibody heavy chain.

Rac activity assay

Rac activity assays were performed as described (27). Briefly, DIV2 hippocampal neurons were stimulated with proNGF (20 ng/ml) for 20 min, and cells were lysed in lysis buffer supplemented with 10 mM MgCl2. Lysates were cleared by centrifugation at 9000g for 1 min, and cleared lysates were incubated with glutathione S-transferase (GST) or GST–PAK-CRIB beads for 30 min at 4°C. Beads were washed, and isolated active Rac was analyzed by Western blot. In parallel, extra lysates were incubated with 1 mM guanosine diphosphate (GDP) or 0.1 mM guanosine 5′-O-(3′-thiotriphosphate) for 30 min at room temperature before incubation with GST–PAK-CRIB beads. Western blots were analyzed by densitometry with ImageJ software, and isolated active Rac was normalized to the input.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/4/202/ra82/DC1

Methods

Fig. S1. Two additional examples showing proNGF-induced growth cone collapse.

Fig. S2. p75NTR and SorCS2 are the receptors mediating growth cone retraction.

Fig. S3. Identification of Trio.

Fig. S4. Generation of the kinase-dead Trio mutant.

Fig. S5. Decrease of Rac but not RhoA activity induces growth cone collapse.

Fig. S6. Fascin and p75NTR form a complex in embryonic brain lysates.

Movie S1. ProNGF leads to growth cone collapse.

References and Notes

Acknowledgments: We thank V. Neubrand for advice on the Rac/Trio work, A. Anastasia for advice on ProNGF, and G. Schiavo and F. Jeanneteau for critical reading of the manuscript. Several cDNA constructs were provided by laboratories that are acknowledged in the manuscript. Individuals requesting these constructs would be referred to the providing laboratory. Funding: This work was supported by the NIH (NS30687 and NS64114 to B.L.H.; NS21072 and HD23315 to M.V.C.; DK32948 and DA15464 to B.A.E. and R.E.M.; and NS050276 and RR017990 to T.A.N.), European Molecular Biology Organization, and the Human Frontier Science Program (K.D.). Author contributions: K.D., T.K., and D.S.S. performed the experiments and analyzed the data; K.D., R.E.M., B.A.E., T.A.N., M.V.C., and B.L.H. designed the experiments and wrote the paper. Competing interests: Cornell University has an issued patent and pending patent applications related to the work reported herein.
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