Neurotrophin Responsiveness of Sympathetic Neurons Is Regulated by Rapid Mobilization of the p75 Receptor to the Cell Surface through TrkA Activation of Arf6

Abstract

The p75 neurotrophin receptor (p75NTR) plays an integral role in patterning the sympathetic nervous system during development. Initially, p75NTR is expressed at low levels as sympathetic axons project toward their targets, which enables neurotrophin-3 (NT3) to activate TrkA receptors and promote growth. Upon reaching nerve growth factor (NGF) producing tissues, p75NTR is upregulated, resulting in formation of TrkA-p75 complexes, which are high-affinity binding sites selective for NGF, thereby blunting NT3 signaling. The level of p75NTR expressed on the neuron surface is instrumental in regulating trophic factor response; however, the mechanisms by which p75NTR expression is regulated are poorly understood. Here, we demonstrate a rapid, translation independent increase in surface expression of p75NTR in response to NGF in rat sympathetic neurons. p75NTR was mobilized to the neuron surface from GGA3-postitive vesicles through activation of the GTPase Arf6, which was stimulated by NGF, but not NT3 binding to TrkA. Arf6 activation required PI3 kinase activity and was prevented by an inhibitor of the cytohesin family of Arf6 guanine nucleotide exchange factors. Overexpression of a constitutively active Arf6 mutant (Q67L) was sufficient to significantly increase surface expression of p75NTR even in the absence of NGF. Functionally, expression of active Arf6 markedly attenuated the ability of NT3 to promote neuronal survival and neurite outgrowth, whereas the NGF response was unaltered. These data suggest that NGF activation of Arf6 through TrkA is critical for the increase in p75NTR surface expression that enables the switch in neurotrophin responsiveness during development in the sympathetic nervous system.SIGNIFICANCE STATEMENT p75NTR is instrumental in the regulation of neuronal survival and apoptosis during development and is also implicated as a contributor to aberrant neurodegeneration in numerous conditions. Therefore, a better understanding of the mechanisms that mediate p75NTR surface availability may provide insight into how and why neurodegenerative processes manifest and reveal new therapeutic targets. Results from this study indicate a novel mechanism by which p75NTR can be rapidly shuttled to the cell surface from existing intracellular pools and explores a unique pathway by which NGF regulates the sympathetic innervation of target tissues, which has profound consequences for the function of these organs.

Keywords: BDNF; nerve growth factor; neurotrophin-3; p75NTR.

Figure 1.

NGF rapidly increases p75NTR surface expression in sympathetic neurons. The surface expression of p75NTR in sympathetic neurons was quantified by immunolabeling live neurons with an antibody to the extracellular domain of the receptor and fluorescently conjugated cholera toxin B to define the cell surface, followed by fixation and immunostaining for the neuron-specific marker TuJ1 or neurofilament. A, Representative images of p75NTR expression in neurons cultured in KCl (12.5 mm) (white circled neuron, top row), treated for 2 h with NGF (40 ng/ml) alone or together with cycloheximide (100 ng/ml) or treated with NT-3 (100 ng/ml). A′, Immunostaining for p75NTR in permeabilized neurons cultured in KCl, revealing the presence of intracellular p75NTR. B, C, The level of p75NTR fluorescence intensity following treatment with NGF for the indicated time was determined by confocal imaging and quantified using Fiji software. D, The expression of p75NTR at the cell surface in response to 2 h exposure to NGF was compared with treatment with NT3 (100 ng/ml) by quantification of fluorescence intensity. E, The total level of p75NTR expression was not altered after 2 h treatment with NGF or NT3, as quantified after permeabilizing the neurons and immunolabeling the receptor. F, Western blot analysis of total neuronal p75NTR expression with or without 2 h NGF treatment. The signal was quantified and normalized to tubulin (n = 3). *p ≤ 0.05, compared with 0 NGF. ***p ≤ 0.001, compared with 0 NGF. ∧p ≤ 0.05 compared with 24 h NGF. Scale bar, 10 μm. G, Representative images of sympathetic neuron growth cones pretreated for 1 h with 10 μm CHX followed by KCl, NGF, or NGF + CHX for 2 h before staining for p75NTR in unpermeabilized conditions. CTB was used to show the growth cone membrane. H, Quantification of p75NTR surface intensity in pixels per area of growth cone (n = 3). *p < 0.05.

Figure 2.

Rapid regulation of neurotrophin receptor expression by NGF is specific for neuronal p75NTR. A, Expression of p75NTR on the surface of satellite glial cells present in cultures of sympathetic neurons grown in KCl (12.5 mm) was detected by immunolabeling p75NTR in live cells with an antibody to the extracellular domain of the receptor. The cell surface was defined using fluorescently labeled CTB. The glial cells maintain robust p75NTR surface expression (white arrow), whereas sympathetic neurons in the same culture exhibit very low expression of p75NTR on the cell surface (white asterisks). The surface level of TrkA (B) or sortilin (C) following treatment with NGF (40 ng/ml) with or without CHX (100 ng/ml) for 0, 1, or 2 h was determined by immunostaining live neurons with an antibody to the extracellular domain of each receptor. The level of receptor at the cell surface was determined by confocal imaging and fluorescence intensity quantified using Fiji software (representative images are shown on the right). There was no significant change in the surface expression of TrkA or sortilin following NGF treatment, with or without CHX (n = 2 or 3). Scale bars, 10 μm.

Figure 3.

NGF induces activation of Arf6. A, NGF was withdrawn from sympathetic neurons and replaced with KCl for ∼18 h; then neurons were exposed to NGF (40 ng/ml) for the indicated time, lysed, and the activated Arf6 determined by pulldown of the GTP bound form with GGA3-coupled beads and Western blot for Arf6 (IP). The lysates were also blotted for total Arf6 (Lys) and the ratio of the active to total used to quantify the fraction of active Arf6, which was expressed as a percentage of neurotrophin naive control. NGF induced a significant increase in GTP-bound Arf6 after 15 min that began to abate after 30 min. B, The activation of Arf6 in the neurons was specific for NGF, as 15 min treatment with BDNF (100 ng/ml) or NT3 (100 ng/ml) did not alter the activity of Arf6 (n = 3–5). **p ≤ 0.01, compared with 0.

Figure 4.

Arf6 regulates p75NTR surface expression. Sympathetic neurons were electroporated with a plasmid expressing GFP (top) or GFP and Q67L Arf6 (bottom) or the Arf6 GAP, ACAP1, then cultured with KCl or NGF. After 3 d, the surface expression of p75NTR in sympathetic neurons was quantified by immunolabeling live neurons with an antibody to the extracellular domain of the receptor, followed by fixation, confocal imaging, and measurement of fluorescence intensity using Fiji software. To define the surface of the cells, fluorescently labeled CTB was added to live neurons during immunostaining. A, Representative images of p75NTR expression on the cell surface in neurons cultured in KCl after electroporation with a vector expressing GFP or GFP and Q67L Arf6. Note the absence of p75NTR in GFP controls (white circle) but increased surface expression in the presence of Q67L Arf6. B, Quantification of p75NTR surface expression in neurons cultured with KCl (white bars) or 40 ng/ml NGF (black bars). The signal is expressed as a percentage relative to neurons expressing GFP alone (n = 5–7). *p ≤ 0.05, compared with GFP electroporated controls. Scale bar, 10 μm.

Figure 5.

NGF upregulates p75NTR surface expression through a TrkA-PI3 kinase-Arf6 pathway. Sympathetic neurons were treated with 40 ng/ml NGF for the indicated time in the absence or presence of (A) the Trk inhibitor K252a (200 nm), (B) the PI3 kinase inhibitor LY294002 (50 μm), or (C) the Arf6 GEF inhibitor SecinH3 (50 μm). The level of p75NTR surface expression was evaluated by immunostaining in unpermeabilized cells; and after fixation, the fluorescence intensity was visualized by confocal imaging and quantified using Fiji software and expressed as a percentage relative to neurons cultured in KCl in the absence of NGF. To define the surface of the cells, fluorescently labeled CTB was added to live neurons during immunostaining (representative images are shown on the right). D, Cell surface p75NTR was also quantified by biotinylation assay. Neurons were cultured in KCl, then treated with NGF with or without SecinH3 (SecH3, 50 μm) for 2 h, then placed at 4°C, and the surface proteins biotinylated. The neurons were then lysed and biotinylated proteins precipitated with streptavidin beads and immunoblotted for p75NTR. To control for nonspecific pulldown of p75NTR, the biotinylation reagent was omitted in one sample (no bio). The band intensity was quantified and normalized to the level of p75NTR in the lysate and expressed as a percentage of KCl control (n = 3–8). *p ≤ 0.05, compared with control. ^#p ≤ 0.05, compared with time-paired inhibitors. ∧p ≤ 0.001, compared with time-paired inhibitors.

Figure 6.

The fraction of intracellular p75NTR colocalized with GGA3 is reduced following NGF treatment. Neurons were electroporated with a plasmid-expressing GGA3 fused to GFP and maintained in KCl for 16 h. Neurons were then either incubated in 40 ng/ml NGF for 2 h or kept in KCl (12.5 mm). High-resolution z stacks of GFP⁺, NF⁺ neurons were acquired using Zeiss 880 LSM with Airyscan (A, A′). Representative images of a neuron maintained in KCl and showing p75NTR (red) colocalized with internal GGA3-GFP (green). Arrows indicate regions of colocalization adjacent to the membrane (inset). B, B′, This colocalization decreases upon NGF treatment. While GGA3 puncta are still visible adjacent to the membrane, most p75NTR has translocated to the surface. Graphs represent quantification of Pearson's r coefficient for p75NTR colocalization with GGA3 (C) or Rab11 (D) from each treatment (n = 3 independent experiments). *p < 0.05. Scale bar, 5 μm.

Figure 7.

Constitutively active Arf6 alters NT3, but not NGF, mediated survival and neurite outgrowth of sympathetic neurons. Sympathetic neurons were electroporated with a plasmid expressing GFP (triangles) or GFP and Q67L Arf6 (square) or untreated (circle), then cultured with KCl for 2 d. The neurons were then treated with the indicated concentration of (A) NGF or (B) NT3 or maintained in KCl. After 48 h, the neurons were fixed, immunostained for TuJ1 to mark neurons, and the nuclei labeled with DAPI. The percentage of apoptotic neurons was determined based on the DAPI staining (n = 3). ***p ≤ 0.01. Neurite outgrowth was analyzed after 16 h of neurotrophin treatment, and neurofilament (NF) immunostaining was used to mark neurites. The longest neurite from each GFP⁺ cell was measured. C, Representative traces of neurite outgrowth from each condition and (D) quantification of GFP⁺ cells in each condition (n = 3 independent experiments). One-way ANOVA between treatments: *p < 0.05 (Tukey's multiple comparisons); GFP NT3 versus Q67L NT3: *p < 0.05; Q67L NGF versus Q67L NT3: *p < 0.05. E, Sympathetic neurons from wild-type or p75NTR^−/− mice were electroporated with Q67L Arf and GFP and immediately plated in either NGF or NT3 for 48 h before fixing. The longest neurite from each GFP⁺, NF⁺ cell was measured (n = 3 independent experiments). One-way ANOVA between treatments: *p < 0.05 (Tukey's multiple comparisons); WT NGF versus WT NT3: **p < 0.01; WT NT3 versus p75KO NT3: *p < 0.05.

Figure 8.

Schematic of rapid NGF regulation of p75NTR trafficking by TrkA activation of Arf6. During axonal growth, sympathetic neurons respond to NT3 through binding to TrkA receptors (1, orange receptors) before reaching NGF-producing target organs. During this time, Arf6 is largely inactive (GDP-bound), and the p75NTR produced is stored in intracellular pools. Once sympathetic axons reach NGF-producing targets, NGF binding to TrkA receptors (2) initiates activation of PI3K (3), which recruits Arf6 GEFs that stimulate GTP binding of Arf6 (4, 5). In its active, GTP-bound state, Arf6 initiates transport of p75NTR from GGA3-containing vesicles to the cell surface (6) and promotes the formation of TrkA-p75NTR complexes (7). The high-affinity TrkA-p75 complexes are selective for NGF. NT3 is no longer able to activate TrkA, and sympathetic neurons become dependent on NGF for survival.