Suppression of neurite outgrowth by high-dose nerve growth factor is independent of functional p75NTR receptors

Abstract

We have previously demonstrated that high concentrations of nerve growth factor suppress neurite outgrowth from sensory neurons. Inhibition could be mediated by either the p75NTR or TrkA receptor. We used a functional block of p75NTR by REX antibody in rat dorsal root ganglion neurons and dorsal root ganglion cultures from p75NTR knockout mice. In both systems, high-dose NGF inhibited neurite outgrowth, implying that p75NTR is not involved in suppression of neurite outgrowth. Confocal images of dissociated dorsal root ganglion neurons exposed to fluorescence-tagged NGF showed ligand internalization. Radioligand binding indicated disappearance of high-affinity binding sites from the surface of dorsal root ganglia after treatment with 200 ng/ml NGF for 1 h. Downstream signaling showed sustained hyperphosphorylation of MAPK (Erk_1–2) but not of SNT or Akt. High-dose NGF may induce cytoplasmic relocation of the receptor TrkA and axonal growth arrest independently of p75NTR.

Introduction

Growth factors of the neurotrophin family, including nerve growth factor (NGF), brain-derived neurotrophin factor (BDNF), and neurotrophin-3 (NT-3), are essential for neuronal survival and differentiation during early stages of development (Sofroniew et al., 2001). NGF neutralizing antibodies given during pregnancy cause loss of sensory and sympathetic neurons in the offspring (Johnson et al., 1980). Gene knockout studies confirm the prominent role of neurotrophins in development Kernie and Parada, 2000, Snider, 1994. NGF in particular has distinct functions in sensory neurons: enabling survival and promoting axonal growth. Hence, NGF was introduced in the clinic with the rationale to enhance neuronal survival and to promote nerve regeneration. Studies on the therapeutic use of NGF have so far met with limited success Apfel, 2001, Quasthoff and Hartung, 2001.

Survival and differentiation are mediated through the NGF receptor TrkA and by distinct transduction pathways Klesse and Parada, 1999, Klesse et al., 1999. The p75NTR receptor is also responsible for survival in embryonic sensory and sympathetic neurons via activation of the transcription factor NFκB Hamanoue et al., 1999, Maggirwar et al., 1998. Conversely, p75NTR induces cell death by activation of the p53/JunK/Fas pathway or by ceramide production in the absence of TrkA (Huang and Reichardt, 2001). Clearly, there is a complex interaction between p75NTR and TrkA (Sofroniew et al., 2001).

NGF-induced signal transduction has been studied in PC12 cells, a NGF-responsive neuronal cell line, and in NGF-dependent primary neurons. Important differences concerning cell survival and differentiation have been recognized between the two cell types. In neurons and in PC12 cells, survival requires activation of the same pathway—the PI3K/Akt cascade. Cell differentiation instead is subserved by at least two different pathways. In PC12 cells, activation of MAPK is necessary and sufficient to induce neurite outgrowth (Klesse et al., 1999). In neurons, MAPK may be involved in neuron extension during development but not during regeneration (Liu and Snider, 2001). In PC12 cells and in cortical neurons, the earliest marker of differentiation is phosphorylation of SNT (suc-associated neurotrophic factor-induced tyrosine phosphorylated target, also called FRS-2 or fibroblast growth factor receptor substrate-2), which occurs within 15 s of cell stimulation. In PC12 cells and in other cell lines, SNT is thought to regulate differentiation by inducing sustained activation of MAPK and neurite growth Kurokawa et al., 2001, Meakin et al., 1999, Xu and Goldfarb, 2001. In neurons, this hypothesis remains to be explored.

In sympathetic and sensory neurons, TrkA binds to NGF to form high-affinity binding sites (K_d ≈ 10⁻¹⁰–10⁻¹¹ M); low-affinity binding sites are formed by binding of NGF to p75NTR (K_d ≈ 10⁻⁸–10⁻⁹ M) Meakin and Shooter, 1992, Sutter et al., 1979. BDNF and NT-3 bind to the p75NTR receptor with the same affinity as NGF. Two different receptors for the same neurotrophin may allow fine-tuning of biological effects through graded concentrations of ligand.

We (Conti et al., 1997) demonstrated that NGF concentrations activating both NGF receptors do not result in cell death but in arrest of neurite growth. In principle, the inhibitory effects of NGF could be mediated by either TrkA or p75NTR. An argument against a role for p75NTR is our earlier finding that outgrowth persists despite high levels of the neurotrophins BDNF and NT-3. To investigate this issue further, we addressed two questions. (1) Which neurotrophin receptor is responsible for inhibiting growth? (2) Does NGF trigger loss of that receptor from the cell surface? Using p75NTR blocking antibodies and primary neurons from p75NTR−/− knockout mice, we examined effects on axonal growth. Using fluorescence cytochemistry and ¹²⁵I-labeled NGF binding, we evaluated receptor abundance and location. TrkA downstream signaling was explored by Western blot. Our results indicate that neurite growth inhibition due to high-dose NGF persisted in the absence of a functional p75NTR receptor. TrkA relocated from the neuronal cell surface to the cytoplasm. Phosphorylation levels of MAPK showed a marked and sustained increase after high-dose NGF exposure, with no corresponding changes in SNT. Neuronal survival was maintained throughout the high-dose NGF treatment.