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An NGF-TrkA-Mediated Retrograde Signal to Transcription Factor CREB in Sympathetic Neurons

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Science  22 Aug 1997:
Vol. 277, Issue 5329, pp. 1097-1100
DOI: 10.1126/science.277.5329.1097

Abstract

Nerve growth factor (NGF) is a neurotrophic factor secreted by cells that are the targets of innervation of sympathetic and some sensory neurons. However, the mechanism by which the NGF signal is propagated from the axon terminal to the cell body, which can be more than 1 meter away, to influence biochemical events critical for growth and survival of neurons has remained unclear. An NGF-mediated signal transmitted from the terminals and distal axons of cultured rat sympathetic neurons to their nuclei regulated phosphorylation of the transcription factor CREB (cyclic adenosine monophosphate response elementbinding protein). Internalization of NGF and its receptor tyrosine kinase TrkA, and their transport to the cell body, were required for transmission of this signal. The tyrosine kinase activity of TrkA was required to maintain it in an autophosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus, an NGF-TrkA complex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons.

The growth and survival of many populations of neurons depends on trophic support provided by their target tissue (1). NGF is secreted by targets of sympathetic and some sensory neurons, and it is also expressed within discrete regions of the central nervous system (1, 2). NGF belongs to a family of structurally related neurotrophic factors termed neurotrophins; this family includes brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) (2). Two cell surface receptors for NGF have been identified: a receptor tyrosine kinase, TrkA, and the low-affinity neurotrophin receptor, p75NTR. NGF exerts its growth- and survival-promoting effects on neurons through activation of TrkA and subsequent biochemical events that ultimately influence the expression of various genes, including those encoding ion channels, neurotransmitter-synthesizing enzymes, and cytoskeletal components (3).

NGF stimulates dimerization and autophosphorylation of TrkA and initiation of intracellular signaling cascades that propagate the signal to the nucleus (4). One transcription factor that is a key target of an NGF-stimulated signaling pathway is CREB (5). Upon exposure of pheochromocytoma-derived cell line PC12 to NGF, CREB becomes phosphorylated on its transcriptional regulatory site Ser133 (5), and this phosphorylation event promotes NGF activation of transcription of the immediate early gene c-fos. Because many NGF-regulated immediate early genes and delayed-response genes contain CREB binding sites within their upstream regulatory regions (5), CREB is likely to be a mediator of the general nuclear response to neurotrophins.

Because NGF is internalized and retrogradely transported from the axon terminal to the cell body (6), NGF itself may carry signals from the axon terminal to the nucleus. Alternatively, TrkA or p75NTR, an NGF-receptor complex, or a terminally derived second messenger molecule might serve as a retrograde messenger (7). To address questions of retrograde NGF signaling, we used compartmentalized cultures of sympathetic neurons (8) and antibodies that distinguish between the Ser133-phosphorylated and unphosphorylated states of CREB (anti–P-CREB) (9) and TrkA (anti–P-Trk) (Fig. 1A). In these cultures, the cell bodies are separated from the axon terminals and distal processes by a distance of either 1 mm or 3 to 4 mm, and the cell bodies and distal processes are located in separate fluid compartments (Fig. 1B). This system enables us to expose isolated terminals and distal axonal processes to NGF and then to assess by immunocytochemistry the phosphorylation state of CREB Ser133 and TrkA in cell bodies.

Figure 1

Phosphorylation of CREB Ser133 after application of NGF to axon terminals and distal processes of sympathetic neurons. (A) Protein immunoblot of extracts of sympathetic neurons. Sympathetic neurons were incubated for 15 min with or without NGF (200 ng/ml) (CTR, control). Whole cell lysates were prepared and protein immunoblot analysis was done with anti–P-CREB (9), anti–P-Trk (16), or anti–tyrosine hydroxylase (TH). CREB is 43 kD, TrkA is 140 kD, and TH is 58 kD. The lower migrating band detected in the P-Trk blot may be a proteolytic fragment of TrkA. (B) Schematic representation of compartmentalized cultures of sympathetic neurons. In center-plated chambers, axons of sympathetic neurons project beneath a Teflon divider that is at least 1 mm wide, whereas in side-plated chambers, the distance between axon terminals and distal processes and the cell bodies is at least 3 mm. (C) Phosphorylation of CREB Ser133 within nuclei of sympathetic neurons after application of NGF to axon terminals and distal processes. Axon terminals and distal processes of sympathetic neurons grown in center-plated chambers were incubated in medium with or without NGF (200 ng/ml) for 20 min. Immunocytochemistry was done with anti–P-CREB, which recognizes CREB that is phosphorylated on Ser133 but not CREB that is unphosphorylated on this residue (9). The percentage of neurons that had nuclei stained with anti–P-CREB was determined by two individuals in blind analyses. Scale bar, 50 μm. (D) Dose-response analysis of NGF induction of CREB phosphorylation. Cell bodies (squares) or axon terminals and distal processes (circles) were treated with the indicated concentrations of NGF for 10 min (cell bodies) or 20 min (axon terminals and distal processes). Cells were then fixed and anti–P-CREB immunocytochemistry was performed. Values are means ± SEM of three independent experiments. (E) Kinetics of NGF induction of phosphorylation of CREB Ser133 in sympathetic neurons. Cell bodies of neurons grown in center-plated chambers (squares), terminals and distal axons of neurons grown in center-plated chambers (circles), or terminals and distal axons of neurons grown in side-plated chambers (triangles) were treated with NGF (200 ng/ml) for the indicated times, and then cells were fixed for immunocytochemistry with anti–P-CREB. Values are means ± SEM of three independent experiments performed in duplicate.

To determine whether NGF induces phosphorylation of CREB Ser133 in sympathetic neurons, we incubated neurons grown in compartmentalized cultures with medium containing a low concentration of NGF (2 ng/ml) for 48 hours. We then exposed either the cell bodies or distal axonal processes to medium containing a high concentration of NGF (200 ng/ml) for various times before fixation and anti–P-CREB immunocytochemistry (10). Exposure of either cell bodies or axon terminals and distal processes to NGF induced phosphorylation of CREB Ser133 within the nuclei of sympathetic neurons (Fig. 1C). Moreover, cell bodies and axon terminals were equally sensitive to NGF (Fig. 1D). However, the kinetics of this NGF-sensitive phosphorylation event differed depending on the site of NGF application or the distance between the cell bodies and the distal processes (Fig. 1E). Application of NGF directly to the cell bodies resulted in phosphorylation of CREB Ser133 within 5 min that returned to the basal level within 40 min. In contrast, upon application of NGF to terminals and distal processes of neurons whose cell bodies were located in center compartments 1 mm away, anti–P-CREB immunoreactivity peaked at 20 min and persisted for at least 1 hour. Furthermore, application of NGF to axon terminals and distal processes at least 3 mm away from the cell bodies resulted in appearance of nuclear P-CREB immunoreactivity that was first detected within 40 min (Fig. 1E). These results indicate that the messenger that transmits the NGF signal from distal axonal processes to CREB within the nucleus travels at a rate of approximately 2 to 4 mm/hour. 125I-labeled NGF is retrogradely transported at an equivalent rate (6) or a slightly faster rate in sympathetic neurons (11).

To determine whether internalization and retrograde transport of NGF are required for retrograde signaling to CREB within the nucleus, we prepared NGF that was covalently coupled to 1 μm–diameter microspheres (12). The NGF-coupled beads, but not control beads (12), induced autophosphorylation of TrkA (Fig. 2A) (13) and activation of the Ras-dependent protein kinase MAPK (mitogen-activated protein kinase) in PC12 cells and in sympathetic neurons (14). The NGF-coupled beads were not internalized by axon terminals and distal processes of sympathetic neurons, nor were they transported to cell bodies (14). Therefore, we used the NGF-coupled beads to determine whether activation of TrkA in terminals was sufficient for signaling to the cell body, or whether internalization and retrograde transport of NGF were also required.

Figure 2

Internalization and retrograde transport of NGF are critical for retrograde signaling to CREB. (A) Phosphorylation of TrkA induced by NGF-coupled beads, but not control (CTR) beads, in PC12 cells. PC12 cells were treated with control medium, control beads (4 μl/ml), NGF (100 ng/ml), or NGF-coupled beads (4 μl/ml) for 10 min (12). Then, TrkA was immunoprecipitated using anti-panTrk (13,22) and immunoblotted with anti-phosphotyrosine as described (22). (B) Internalization and retrograde transport of NGF are required for phosphorylation of CREB Ser133. Axon terminals and distal processes of sympathetic neurons grown in center-plated chambers were treated with control medium, soluble NGF (200 ng/ml), NGF-coupled beads (4 μl/ml), or control beads for 20 min. Alternatively, cell bodies of neurons grown in center-plated chambers were treated with the same stimuli for 10 min. Cells were then fixed for immunocytochemistry with anti–P-CREB. Values are means ± SEM of three independent experiments performed in duplicate.

Upon exposure of cell bodies of sympathetic neurons to either soluble NGF or NGF-coupled beads, phosphorylation of CREB Ser133 was detected in nuclei of nearly 80% of the neurons; this result was not seen with control beads (Fig. 2B). In contrast, NGF-coupled beads failed to stimulate CREB phosphorylation when applied to axon terminals and distal processes of sympathetic neurons (Fig. 2B). However, in parallel cultures, soluble NGF stimulated CREB phosphorylation in nearly 80% of neurons when applied to axon terminals. Thus, although internalization of NGF is not necessary for propagation of the NGF signal from the plasma membrane of the cell body to the nucleus, internalization and retrograde transport of NGF are critical for propagation of the NGF signal from the axon terminal and distal process to the nucleus. These results support a model in which NGF itself is a critical component of the retrograde signaling complex.

The possibility that NGF is retrogradely transported to the cell body of sympathetic neurons as part of a complex with one of its receptors, TrkA, is supported by the observation that NGF remains associated with tyrosine-phosphorylated TrkA in internalized vesicles purified from NGF-treated PC12 cells (15). We therefore tested whether exposure of terminals of sympathetic neurons to soluble NGF resulted in retrograde transport of autophosphorylated TrkA receptors. Appearance of tyrosine-phosphorylated TrkA (P-Trk) in cell bodies was determined by immunocytochemistry with antibodies that recognize TrkA only when it is phosphorylated on two tyrosine residues, Tyr674and Tyr675 (Fig. 1A) (16). Upon exposure of axon terminals and distal processes to NGF, the amount of P-Trk immunoreactivity was increased in the distal processes and also in cell bodies (Fig. 3), which suggests that P-Trk, like NGF, is retrogradely transported in sympathetic neurons. This conclusion is consistent with the observation that P-TrkA accumulates on the distal side of a ligation (17) or crush (18) of the sciatic nerve. Because NGF and TrkA remain associated within internalized vesicles (15), these results support a model in which NGF maintains cotransported TrkA in an active state.

Figure 3

Appearance of tyrosine-phosphorylated TrkA in distal processes and cell bodies after application of NGF to axon terminals and distal processes. Axon terminals and distal processes of neurons grown in center-plated chambers were untreated (control) or were treated with NGF (200 ng/ml) for 20 min. In the lower panels, the cell bodies were first treated with K-252a (100 nM, 30 min) and the terminals were then treated with NGF. Cells were fixed for immunocytochemistry with anti–P-Trk (Fig. 1A) (16). This experiment was performed three times with similar results. Incubation of anti–P-Trk with the phosphotyrosine-containing Trk peptide used to generate the antibody, but not control peptides, abolished P-Trk immunoreactivity (14). Scale bar, 50 μm.

To test this idea, we used a potent and selective inhibitor of Trk protein kinase activity, K-252a (19, 20). Application of K-252a to cell bodies prevented the appearance of P-Trk in cell bodies after exposure of axon terminals and distal processes to NGF (Fig. 3). In contrast, application of K-252a to cell bodies did not block NGF-induced accumulation of P-Trk immunoreactivity in distal axons and terminals. Thus, tyrosine kinase activity of retrogradely transported TrkA is critical for maintaining the receptor in an autophosphorylated state upon its arrival in the cell body.

We next tested the possibility that retrogradely transported, catalytically active TrkA contributes to retrograde signaling to the nucleus. For these experiments, we assessed the ability of terminally applied NGF to induce CREB phosphorylation in neurons in which we inhibited TrkA kinase activity in cell bodies but not in axon terminals and distal processes. When applied to cell bodies, K-252a blocked phosphorylation of CREB Ser133 in response to application of NGF to axon terminals and distal processes (Fig. 4). In contrast, K-252a treatment of axon terminals and distal processes did not block phosphorylation of CREB Ser133 in response to application of NGF directly to the cell bodies. We conclude that retrogradely transported, catalytically active TrkA and its ligand, NGF, are components of a complex that conveys the NGF signal from the axon terminals to CREB within the nuclei of sympathetic neurons.

Figure 4

Requirement of tyrosine kinase activity of retrogradely transported TrkA for propagation of the retrograde signal to CREB. Sympathetic neurons grown in center-plated chambers were untreated, or either cell bodies or axon terminals and distal processes were exposed to K-252a (100 nM, 30 min). Then, NGF was applied to axon terminals and distal processes or cell bodies, and P-CREB immunocytochemistry was done with anti–P-CREB. Values are means ± SEM of three independent experiments performed in duplicate.

  • * These authors contributed equally to this report.

  • To whom correspondence should be addressed. E-mail: david_ginty{at}qmail.bs.jhu.edu

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