We have discovered that axotomy of sciatic nerve induces Schwann cells distal to the lesion to express de novo, or at greatly increased levels, receptors for nerve growth factor (NGF). Surgical transection of sciatic nerve was performed on adult Sprague-Dawley rats, and, at various times after the operation, the following tissues were dissected for quantitation of NGF receptor: L4 and L5 dorsal root ganglia, sciatic nerve proximal to the transection, sciatic nerve distal to the transection, tibialis anterior muscle, and skin of the dorsum of the foot. The NGF receptor content of these samples was determined by labeling receptor molecules with radioiodinated NGF (1251-NGF) and then specifically immunoprecipitating the 125I-NGF-receptor complexes with 192-IgG, a monoclonal antibody directed against the rat NGF receptor. We observed a time-dependent increase in the amount of radioligand-labeled NGF receptor proteins found in the distal segment of transected sciatic nerve; by 7 days the density of receptor (crosslinked '251-NGF molecules per mg of homogenate protein) had increased at least 50-fold. The number of receptor molecules in tibialis anterior muscle and dorsal foot skin, two structures denervated by the transection, also increased, with time courses parallel to that of distal sciatic nerve. There was little increase in the density of NGF receptors in the sciatic nerve proximal to the lesion and in the dorsal root ganglia. Immunohistochemical examination of the distal portion of transected sciatic nerve and of the muscle, with 192-IgG as the primary ligand, revealed prominent and exclusive staining of apparently all Schwann cells of the endoneurium, indicating that these peripheral neuroglial cells were expressing NGF receptors. These results show that axonal damage can induce the expression of NGF receptors in the population of sheath cells thought to promote neuronal regeneration. This dramatic increase in NGF receptors may be a mechanism to facilitate the regeneration of NGF-responsive neurons.Nerve growth factor (NGF) is a polypeptide neurotrophic agent that promotes the survival and function of sympathetic neurons and neural crest-derived sensory neurons (1). NGF also exerts chemotactic, or tropic, activity on sensory neurons (2, 3). These biological actions of NGF are initiated by the specific binding of the protein to cell-surface receptor molecules (4). In addition to sympathetic and sensory neurons, the NGF receptor has been found on several nonneuronal cells, including the clonal rat pheochromocytoma PC12 cells (5) and the A875 clonal line of human melanoma (6). Schwann cells, the peripheral neuroglial cells that ensheath nerve axons, also express NGF receptors when placed into tissue culture during a restricted period of embryogenesis (7-11); a subpopulation of Schwann cells from cultured embryonic chicken dorsal root ganglia (DRG) displays specific binding of NGF during embryonic days 6-12 (8). NGF receptors have not been detected on Schwann cells in vivo, and the function of the recept...

Axotomy of sciatic nerve fibers in adult rats induces expression of NGF receptor in the entire population of Schwann cells located distal to the injury (Taniuchi et al., 1986b). In the present study we have used immunocytochemistry, with a monoclonal antibody directed against the rat NGF receptor, to examine axotomized peripheral nerves by light and electron microscopy. We have found that (1) the NGF receptor molecules were localized to the cell surface of Schwann cells forming bands of Bungner; (2) axonal regeneration into the distal portion of sciatic nerve coincided temporally and spatially with a decrease in Schwann cell expression of NGF receptor; (3) Schwann cell NGF receptor could be induced by axotomy of NGF-independent neurons, such as motoneurons and parasympathetic neurons; and (4) the presence of axon-Schwann cell contact was inversely related to expression of Schwann cell NGF receptor. Using biochemical assays we have found that, in striking contrast to peripheral nerves, there was no detectable induction of NGF receptor in the spinal cord and brain after axotomy of NGF receptor-bearing fibers. Filtration assays of 125I-NGF binding to the induced NGF receptors of Schwann cells measured a Kd of 1.5 nM and a fast dissociation rate, both characteristics of class II receptor sites. We conclude that Wallerian degeneration induces Schwann cells, but not central neuroglia, to produce and position upon their plasmalemmal surface the class II NGF receptor molecules. The induction is ubiquitous among Schwann cells, irrespective of the type of axon they originally ensheathed. Expression of Schwann cell NGF receptor is negatively regulated by axonal contact, being induced when axons degenerate and suppressed when regenerating axons grow out along the Schwann cell surface. We propose that the induced NGF receptors function to bind NGF molecules upon the Schwann cell surface and thereby provide a substratum laden with trophic support and chemotactic guidance for regenerating sensory and sympathetic neurons.

Previously, we reported that nerve growth factor (NGF) is required to maintain herpes simplex virus (HSV) latency in cultures of rat sympathetic neurons (Wilcox and Johnson, 1987, 1988). Here, we extend these results by showing that NGF was also required to maintain HSV latency in cultures of sensory neurons obtained from dorsal root ganglia of rats, monkeys, and humans. The interruption of the neuronal supply of NGF for 1 hr reactivated HSV, indicating that the latent virus was exquisitely sensitive to perturbations in the concentration or binding of NGF. A species-specific monoclonal antibody directed against the human NGF-receptor, which blocks NGF binding, reactivated latent HSV in human, but not rat, sensory neurons. In contrast, a monoclonal antibody against the rat NGF-receptor, which binds the receptor without blocking NGF action, did not produce reactivation. These results indicate that the effects of NGF on HSV latency are mediated via NGF binding to the NGF receptor. In addition, treatments that interfere with specific steps in the transduction of the NGF signal, including treatment with 6-hydroxydopamine and colchicine, reactivated latent HSV. Further, in neurons harboring latent virus, interruption of protein synthesis or RNA transcription for 1 hr resulted in viral reactivation, suggesting that a short-lived factor may be present in neurons which represses viral reactivation.

NGF acts on responsive neurons by binding to specific NGF receptors on axonal termini, after which a critical biochemical signal is retrogradely transported to the cell body. The identity of the signal(s) is unknown; candidates include NGF itself or some other "second messenger." A possible second messenger is the NGF receptor. As a first step in assessing the possible role of NGF receptor in the generation of the NGF-dependent signal, and in understanding the economy of NGF receptor synthesis and utilization, we determined whether the NGF receptor is retrogradely transported. Using immunohistochemical staining with a monoclonal antibody (192-IgG) against rat NGF receptor, we looked for accumulation of NGF receptor molecules distal (retrograde transport), as well as proximal (anterograde transport), to sites of axonal ligation or transection. By 10-12 hr in both the ligated sciatic nerve and the lesioned fimbria-fornix, accumulated NGF receptor was detected proximal and distal to the ligation/lesion site. The transported receptor presumably was located in sympathetic and sensory neurons in the sciatic nerve and in forebrain cholinergic neurons projecting from the medial septum to the hippocampus. In both anatomical sites, accumulation of NGF receptor on the proximal (anterograde) side occurred in streams of fine axonal processes, whereas staining on the distal (retrograde) side occurred in varicose or granular configurations. These results raise the possibility that the NGF receptor has a role in the mechanism of NGF beyond the initial binding event at the plasma membrane of the axonal terminus.

Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis. In this paper we report that AraC kills postmitotic rat sympathetic neurons in a fashion similar to the neuronal death that follows nerve growth factor (NGF) deprivation. Postmitotic rat sympathetic neurons were cultured for 1 week in the presence of NGF and then treated with AraC, still in the presence of NGF. AraC killed neurons after 4 d with an EC50 of 50 microns. The morphological and temporal characteristics of neuronal death that began around 3 d after addition of AraC were indistinguishable from those observed beginning 24 hr after NGF deprivation. Death caused by AraC was prevented by the same agents that prevent the death of NGF-deprived neurons, which included inhibitors of RNA and protein synthesis, a cAMP analog, and depolarizing concentrations of KCl. In contrast, neuronal death caused by ultraviolet irradiation, ricin toxin, and a variety of other toxic insults did not share these morphological, biochemical, or temporal characteristics. Other antimitotic drugs, including adenine arabinoside, thymine arabinoside, fluorodeoxyuridine, hydroxyurea, and aphidicolin, did not kill neurons. AraC caused neurons to behave as if deprived of NGF by interfering with deoxycytidine (dC) metabolism distinct from DNA biosynthesis. dC entirely prevented the neurotoxicity of AraC, even when present at a concentration 1000-fold less than that of AraC. Other deoxynucleosides, and cytidine, did not prevent AraC neurotoxicity. dC could not, however, substitute for NGF and thus is unlikely to be a direct mediator of NGF action. It is hypothesized that dC may participate in a pathway, distinct from DNA synthesis, that is necessary for neurons to respond to exogenous trophic factors.